Hormonal Signatures of Exercise: A Comparative Analysis Across Populations for Research and Therapeutics

Abstract

This article provides a comprehensive synthesis of current research on how diverse populations exhibit distinct hormonal responses to various exercise modalities. It explores the foundational neuroendocrine mechanisms, detailing acute and chronic adaptations in axes such as HPA, HPT, and HPG. The content further addresses methodological considerations for measuring these responses in research settings, identifies factors contributing to response variation (e.g., sex, age, training status), and offers comparative analyses across key demographics. Designed for researchers, scientists, and drug development professionals, this review highlights the implications of these differential responses for developing personalized exercise prescriptions and novel therapeutic strategies targeting metabolic and endocrine pathways.

Decoding the Exercise-Endocrine Axis: Fundamental Mechanisms and Key Hormonal Players

Neuroendocrine Exercise Response Model (HERM)

The Hormonal Exercise Response Model (HERM) provides a conceptual framework for understanding the endocrine system's reactivity to the physical stress of exercise [1]. This model organizes the complex hormonal changes during physical activity into an interactive, multi-phase process, illustrating how the body transitions from neural-driven to humorally-controlled responses as exercise duration increases [1] [2]. For researchers investigating hormonal responses across different populations, HERM offers a structured approach to conceptualizing how exercise volume, intensity, and individual characteristics shape endocrine adaptations.

The HERM framework is particularly valuable for contextualizing research comparing hormonal exercise responses across diverse populations, as it accounts for the temporal sequence of endocrine events and the shifting regulatory mechanisms that occur during sustained physical activity [1].

The Three-Phase HERM Framework

Phase I: Immediate Neural Response

The initial phase begins within seconds of exercise onset and is predominantly driven by neural mechanisms [1]. This response involves:

• Sympathetic Nervous System Activation: Increased sympathetic outflow leads to catecholamine release (norepinephrine) directly at target tissues and through sympathetic "spillover" into circulation [1].
• Adrenal Medullary Activation: The sympathetic-adrenal connection stimulates the adrenal medulla to release additional catecholamines (epinephrine > norepinephrine) into circulation [1].
• Pancreatic Hormone Modulation: Insulin secretion becomes inhibited while glucagon secretion is stimulated [1].
• Regulatory Mechanism: This phase operates primarily through feed-forward mechanisms of the central nervous system, modified by peripheral afferent neural input from sensory receptors in skeletal muscle [1].

Phase II: Intermediate Pituitary Response

The intermediate phase develops within minutes of exercise onset and represents a transition between neural and hormonal dominance [1]. Key aspects include:

• Hypothalamic Activation: The hypothalamus begins releasing factors including thyrotropin-releasing factor, corticotrophin-releasing factor (CRF), and growth hormone-releasing factor [1].
• Pituitary Engagement: The anterior pituitary gland responds to hypothalamic stimuli by releasing trophic hormones into circulation [1].
• Peripheral Endocrine Activation: Pituitary hormones stimulate specific peripheral target glands to release additional hormones [1].
• Exemplary Pathway: The hypothalamic-pituitary-adrenal cortical axis demonstrates this phase, where CRF stimulates adrenocorticotrophic hormone release, which ultimately triggers cortisol release from the adrenal cortex [1].

Phase III: Prolonged Humoral Adjustment

During extended exercise sessions, the response transitions to the third phase characterized by humoral and hormonal dominance [1]. This phase features:

• Sympathetic-Augmentation: Sympathetic-adrenal axis responses are augmented by hormones from the anterior and posterior pituitary (growth hormone, prolactin, antidiuretic hormone) and peripheral endocrine glands (testosterone, thyroxine, triiodothyronine, insulin-like growth factor-1) [1].
• Fluid Regulation: As fluid shifts occur due to sweating and vascular changes, the renin-angiotensin-aldosterone system (RAAS) activates to induce vasoconstriction and water resorption at the kidneys [1].
• Cytokine Involvement: Skeletal muscle begins releasing cytokines (e.g., interleukin-6) that affect other hormone releases and signal energy substrate mobilization and immune responses [1].
• Regulatory Shift: This phase demonstrates a transition toward feedback control mechanisms rather than feed-forward regulation, with humoral and hormonal stimuli becoming increasingly influential as exercise continues [1].

Table 1: Key Characteristics of HERM Phases

Phase	Timeframe	Primary Drivers	Key Hormones Involved	Regulatory Mechanism
Phase I: Immediate	Seconds	Neural mechanisms	Catecholamines (epinephrine, norepinephrine)	Feed-forward control
Phase II: Intermediate	<1 minute	Neural-pituitary interplay	Releasing factors, trophic hormones, cortisol	Transitional
Phase III: Prolonged	Extended exercise	Humoral factors	GH, prolactin, ADH, testosterone, cytokines	Feedback control

Experimental Methodologies for HERM Investigation

Population Selection Considerations

When designing studies to investigate HERM across different populations, researchers must account for numerous variables that significantly modify hormonal responses [2]:

• Genetic background: Ethnic variations in endocrine reactivity
• Demographic factors: Age, sex, and stage of sexual development
• Training status: Level of fitness, training history, and conditioning
• Physiological conditions: Nutritional status, energy availability, biological rhythms
• Lifestyle factors: Drug/supplement intake, sleep patterns, stress levels
• Health status: Previous or current pathologies that may affect endocrine function

Standardized Exercise Protocols

To ensure comparable results across population studies, researchers should implement standardized exercise protocols with careful attention to:

• Exercise modality: Type of exercise (resistance, endurance, high-intensity interval training)
• Intensity quantification: Percentage of VOâ‚‚ max, heart rate reserve, or one-repetition maximum
• Duration parameters: Time domains for each HERM phase
• Environmental controls: Temperature, humidity, and altitude standardization
• Temporal consistency: Time of day to account for circadian hormonal variations

Hormonal Assessment Methodologies

Accurate measurement of hormonal parameters requires rigorous methodological consistency:

Table 2: Key Hormonal Assessment Methodologies in HERM Research

Hormone Category	Specific Hormones	Sample Type	Assessment Method	Timing Considerations
Catecholamines	Epinephrine, Norepinephrine	Plasma	HPLC, ELISA	Rapid processing required
Hypothalamic-Pituitary	CRF, GHRH, TRH	Plasma	Immunoassays	Low concentrations challenging
Anterior Pituitary	ACTH, GH, TSH	Serum	Chemiluminescence, RIA	Pulsatile secretion patterns
Adrenal Cortex	Cortisol	Serum, Saliva	Immunoassays, LC-MS	Diurnal variation significant
Gonadal Steroids	Testosterone, Estradiol	Serum	LC-MS/MS, Immunoassays	Cyclic variations in females
Pancreatic	Insulin, Glucagon	Plasma	ELISA, RIA	Rapid degradation concerns

Temporal Sampling Protocols

Comprehensive HERM investigation requires strategic temporal sampling to capture phase transitions:

• Baseline sampling: Pre-exercise resting measurements after appropriate stabilization
• Early exercise sampling: 0-10 minutes to capture Phase I responses
• Intermediate sampling: 10-30 minutes to identify Phase II transitions
• Extended sampling: 30+ minutes to characterize Phase III adaptations
• Recovery sampling: Post-exercise to document return to baseline kinetics

Comparative Hormonal Responses Across Populations

The HERM framework reveals significant variations in hormonal exercise responses across different population subgroups. Understanding these differences is crucial for personalized exercise prescription and population-specific training recommendations.

Sex-Based Variations in HERM Responses

Research conducted within the HERM context demonstrates distinct hormonal response patterns between males and females [2]:

Table 3: Sex-Specific Variations in Hormonal Exercise Responses

Hormone	Basal Levels F/M	Acute Exercise Response F/M	Training Adaptation F/M	Population Considerations
Growth Hormone (GH)	↑ Females	⇑ Females	↑/=/↓	Greater response in women
IGF-1	↑ Males	⇑ Males	↑/=/↓	More pronounced in males
Cortisol	↑ Males	⇑ Males-↑ Females	↑/=/↓	Sex-dependent stress response
Testosterone	↑ Males	⇑ Males-↑ Females	=/↓	Anabolic capacity differences
Catecholamines	F = M	↑ Males	↑/=/↓	Sympathetic reactivity variance

Training Status and HERM Adaptations

Training status significantly modifies HERM phase characteristics [2] [3]:

• Trained individuals: Exhibit attenuated hormonal responses during acute exercise at the same absolute intensity
• Untrained individuals: Demonstrate exaggerated hormonal reactivity, particularly in Phase I and II responses
• Overtrained athletes: Show dysregulated HERM patterns, often with elevated cortisol and suppressed testosterone
• Detraining effects: Partial reversal of training adaptations within 2-4 weeks of training cessation

Age-Related Modifications in HERM

Aging progressively alters hormonal exercise responses [2]:

• Phase I: Diminished catecholamine response in older adults
• Phase II: Blunted growth hormone and ACTH release in aged populations
• Phase III: Altered cytokine responses and delayed recovery kinetics
• Overall effect: Attenuated hypothalamic-pituitary axis reactivity in older versus younger individuals

Research Reagent Solutions for HERM Investigation

Table 4: Essential Research Materials for HERM Studies

Research Tool Category	Specific Examples	Application in HERM Research	Technical Considerations
Hormone Assay Kits	ELISA, RIA, CLIA kits	Quantification of specific hormones	Cross-reactivity assessments needed
Chromatography Systems	HPLC, LC-MS systems	Catecholamine measurement	Sensitivity to low concentrations
Automated Blood Samplers	Portable venous catheters	Repeated sampling during exercise	Participant mobility constraints
Biomimetic Binding Assays	AGP, IAM stationary phases	Protein and phospholipid binding studies	Correlation with hormonal activity
Exercise Equipment	Treadmills, cycle ergometers	Standardized exercise protocols	Calibration and verification
Data Analysis Software	Statistical packages	Modeling hormonal response patterns	Handling of repeated measures

HERM Visualization: Signaling Pathways and Experimental Workflows

HERM Three-Phase Response Pathway

HERM Experimental Research Workflow

Implications for Research and Clinical Applications

The HERM framework provides valuable insights for both research design and practical applications:

Research Design Considerations

• Temporal resolution: Studies must account for the different HERM phases with appropriate sampling frequency
• Population stratification: Research cohorts should be stratified based on known HERM modifiers (sex, age, training status)
• Intervention timing: Exercise interventions may have different effects depending on the targeted HERM phase
• Outcome measures: Multiple hormonal markers across different axes are necessary to capture comprehensive HERM responses

Clinical and Performance Applications

• Overtraining monitoring: HERM dysregulation can serve as early detection for overtraining syndrome [2]
• Personalized training: Understanding individual HERM patterns can optimize exercise prescription
• Rehabilitation programming: HERM principles can guide exercise progression in clinical populations
• Hormonal assessment: Strategic timing of hormonal measurements based on HERM phase characteristics

The HERM framework continues to evolve as research reveals additional complexity in endocrine exercise responses. Future investigations incorporating advanced molecular techniques, continuous biomarker monitoring, and multi-omics approaches will further refine our understanding of how different populations transition through the distinct phases of neuroendocrine exercise response.

Physical exercise presents a potent stressor to human physiology, triggering a complex cascade of endocrine responses aimed at restoring homeostasis. These hormonal adjustments can be broadly categorized into two distinct temporal patterns: acute adaptations, which are transient changes occurring during and immediately after a single exercise bout, and chronic adaptations, which represent long-term, stable shifts in basal hormonal levels and system reactivity resulting from repeated training. The Hormonal Exercise Response Model (HERM) provides a framework for understanding this progression, describing a shift from rapid, neural-driven hormone secretion during initial exercise to more refined feedback-driven mechanisms and altered baseline function after sustained training [4]. For researchers and drug development professionals, dissecting these adaptations is critical for designing targeted exercise mimetics, optimizing hormonal therapies, and understanding the pathophysiology of metabolic and stress-related disorders. This guide objectively compares these hormonal responses across different exercise modalities and populations, providing a synthesis of experimental data and methodologies.

Acute Hormonal Adaptations to Exercise

Acute hormonal responses are characterized by rapid, often transient, increases or decreases in circulating hormone levels, directly triggered by the physiological demands of a single exercise session.

The Stress Axis: HPA and Catecholamines

The hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic nervous system are among the first to respond to exercise-induced stress.

• HPA Axis: A single bout of endurance exercise stimulates the HPA axis, leading to increased secretion of corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) from the hypothalamus, which in turn prompts the anterior pituitary to release adrenocorticotropic hormone (ACTH) and β-endorphin [5]. ACTH then drives the release of cortisol from the adrenal cortex. This response is highly dependent on exercise intensity and duration, with a clear threshold required to elicit a significant rise in cortisol [5].
• Catecholamines: The locus caeruleus/norepinephrine (LC/NE) system is activated, leading to a swift increase in circulating catecholamines (epinephrine and norepinephrine) [5]. These hormones are critical for optimizing force production, increasing cardiovascular tone, and liberating energy substrates (e.g., promoting glycogenolysis and lipolysis) [6] [7].

Anabolic and Metabolic Hormones

Simultaneously, anabolic and metabolic hormones are mobilized to support energy production and initiate tissue repair processes.

• Growth Hormone (GH): GH release is potently stimulated by exercise, starting 10-20 minutes after onset and peaking either at the end or shortly after the session [4]. The magnitude of this response is positively correlated with exercise duration and, particularly, intensity [4].
• Testosterone: Resistance exercise has been shown to elicit a significant acute increase in testosterone [6] [8]. Protocols that are high in volume, moderate to high in intensity, use short rest intervals, and stress a large muscle mass tend to produce the greatest acute elevations [6].
• Insulin and Glucagon: As part of the energy mobilization phase, exercise triggers a rapid sympathetic nervous system response that decreases insulin secretion and increases glucagon, facilitating a rise in blood glucose to fuel working muscles [4].

Table 1: Summary of Key Acute Hormonal Responses to a Single Bout of Exercise

Hormone	Direction of Change	Primary Stimulus	Proposed Physiological Role
Cortisol	Increase [5]	Intensity/Duration of Exercise (HPA Axis Activation)	Mobilize energy substrates, modulate inflammation [5]
Catecholamines	Increase [5] [6]	Sympathetic Nervous System Activation	Increase cardiac output, liberate energy (glycogenolysis, lipolysis) [6]
Growth Hormone (GH)	Increase [6] [4]	Exercise Intensity & Duration [4]	Promote lipolysis, support substrate availability and tissue repair [4]
Testosterone	Increase [6]	Resistance Exercise (High volume, moderate-high intensity, short rest) [6]	Mediate anabolic signaling, promote tissue growth and remodelling [6] [8]
Insulin	Decrease [4]	Sympathetic Activation	Reduce glucose uptake in non-working tissues, support blood glucose availability [4]

Chronic Hormonal Adaptations to Exercise

Repeated exposure to exercise leads to chronic adaptations, which are characterized by changes in basal (resting) hormone levels and an altered responsiveness of the endocrine systems to subsequent exercise bouts.

Basal Shifts in the Stress Axis

With chronic training, the body's stress systems undergo significant recalibration, often resulting in a more efficient and attenuated response to a given exercise stimulus.

• HPA Axis: Highly trained athletes demonstrate adaptive changes in basal HPA function. One study found that ultramarathon runners exhibited a phase-shifted diurnal cortisol rhythm and significantly higher basal ACTH levels compared to sedentary controls, yet no overall difference in plasma cortisol, suggesting a blunting of the adrenal cortisol response to chronic exercise stress [9]. Furthermore, regular high-intensity interval exercise (HIIE) has been shown to lower basal cortisol concentrations [5].
• Catecholamines: The catecholamine response to a standard bout of high-intensity exercise is reduced following regular HIIE training, indicating an adaptation of the sympathetic nervous system [5].

Anabolic and Metabolic Recalibration

Chronic exercise also induces stable changes in the anabolic and metabolic environment, which underpin long-term improvements in body composition and metabolic health.

• Anabolic Hormones: After prolonged training, basal levels of hormones like testosterone and GH typically show only marginal changes or slight reductions, constrained by a "basement effect" [4]. The more critical adaptation is an attenuated acute hormonal response to a standard exercise bout after training, reflecting reduced stress reactivity and enhanced sensitivity of target tissues [4].
• Insulin Sensitivity: Regular aerobic exercise is linked to a profound chronic increase in insulin sensitivity within skeletal muscle. This adaptation is facilitated by increased concentrations of GLUT4 transporter proteins, which improve glucose translocation into the muscle [10]. This enhanced insulin sensitivity can persist for 12-48 hours after the last exercise session [10].

Table 2: Summary of Key Chronic Hormonal Adaptations to Regular Exercise Training

Hormone / System	Basal (Resting) Change	Response to Acute Exercise Post-Training	Proposed Physiological Role of Adaptation
HPA Axis (Cortisol)	Phase-shifted rhythm; Blunted adrenal response [9] / Lower basal with HIIE [5]	Altered (e.g., higher ACTH but similar cortisol) [9]	Improved stress management, energy conservation
Catecholamines	Not well defined	Reduced response to standard bout [5]	Increased metabolic efficiency, reduced cardiovascular strain
Growth Hormone (GH)	Marginal change [4]	Attenuated acute spike [4]	Reflects enhanced tissue sensitivity and efficiency
Testosterone	Marginal change [6] [4]	Attenuated acute spike [4]	Reflects enhanced tissue sensitivity and efficiency
Insulin Sensitivity	Marked Improvement [10]	Improved glucose clearance post-exercise	Enhanced metabolic health, reduced risk of Type 2 Diabetes [10]

Comparative Analysis Across Exercise Modalities

The nature of hormonal adaptations is profoundly influenced by the type of exercise performed. The following section details the specific endocrine responses and adaptations to different exercise modalities.

Endurance Exercise

• Acute Response: A single bout stimulates the HPA axis, leading to increased cortisol and β-endorphin, and induces a significant GH peak [5].
• Chronic Adaptation: Regular endurance training can lead to relatively increased basal cortisolemia and an altered diurnal rhythm [5] [9]. It consistently improves insulin sensitivity and demonstrates anti-inflammatory immunoprotective effects [10] [11].

Resistance Exercise

• Acute Response: Characterized by mild HPA axis stimulation and significant acute elevations in anabolic hormones like testosterone and GH, particularly with protocols that are high in volume, use moderate to high intensities (65-85% 1RM), and incorporate short rest intervals [5] [6].
• Chronic Adaptation: Resting hormonal concentrations may not change significantly despite increases in muscle strength and hypertrophy, highlighting the importance of the acute response and mechanical signaling for tissue remodelling [6]. Regular training in older populations is associated with an attenuated inflammatory response and decreased resting cytokine concentrations [5] [11].

High-Intensity Interval Exercise (HIIE)

• Acute Response: A single bout of HIIE induces a significant cortisol increase and a robust GH response [5].
• Chronic Adaptation: Regular HIIE training leads to lowered basal cortisol concentrations and a reduced catecholamine response compared to a single bout, indicating an efficient adaptation of the stress systems [5]. Immunological responses are mixed, often combining transient pro-inflammatory responses with long-term benefits [11].

Table 3: Comparison of Hormonal Responses by Exercise Modality

Modality	Acute Cortisol Response	Acute Anabolic (Test/GH) Response	Chronic Basal Adaptation	Key Health & Performance Links
Endurance	Increase [5]	GH peak [5]	↑ Basal cortisolemia; ↑ Insulin sensitivity [10] [5]	Improved cardiorespiratory fitness, metabolic health [10] [5]
Resistance	Mild Increase [5]	Significant Increase (Test, GH) with specific protocols [6]	Minimal basal hormonal change; ↓ Resting inflammation [5] [6] [11]	Increased muscle strength & hypertrophy [6]
HIIE	Increase [5]	GH peak [5]	↓ Basal cortisol; ↓ Catecholamine response [5]	Time-efficient cardiorespiratory & metabolic improvements [5]

Experimental Protocols for Hormonal Analysis

For researchers seeking to replicate or build upon these findings, a clear understanding of the experimental designs is crucial.

Protocol for Acute Hormonal Response

• Objective: To quantify the transient hormonal changes in response to a single exercise session.
• Population: Typically involves healthy, sedentary to recreationally active adults to observe a clear response. Comparisons are often made between groups (e.g., trained vs. untrained).
• Exercise Stimulus:
  • Endurance: Cycle ergometer or treadmill running at a fixed intensity (e.g., 70-80% VOâ‚‚max) for 30-60 minutes [10] [5].
  • Resistance: 3-6 sets of 8-12 repetitions at 65-85% 1RM for compound exercises like squat and bench press, with short (60-90s) rest intervals [6].
  • HIIE: Repeated bouts (e.g., 6-8) of 60-second efforts at 85-100% VOâ‚‚max, interspersed with 60-90 seconds of active recovery [5].
• Blood Sampling & Analysis: Blood samples are drawn pre-exercise (baseline), immediately post-exercise, and at regular intervals during recovery (e.g., 15, 30, 60, 120 mins). Plasma or serum is analyzed using techniques like radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) for hormones like cortisol, testosterone, GH, and ACTH [9].

Protocol for Chronic Hormonal Adaptation

• Objective: To assess long-term changes in basal hormone levels and the attenuated response to a standardized exercise test.
• Study Design: Longitudinal training study, typically lasting 6 weeks to 12 months [10] [9].
• Training Intervention:
  • Endurance: 3-5 sessions per week of continuous cycling/running at 70-80% VOâ‚‚max for 30-60 minutes per session [10].
  • Resistance: 2-3 sessions per week, focusing on hypertrophy-type protocols (3 sets of 8-12RM on major muscle groups) [6].
  • HIIE: 3 sessions per week of a protocol similar to the acute stimulus described above [5].
• Testing Points: Basal (resting) blood samples are taken after a 48-72 hour period without exercise at the start and end of the training period. Additionally, the acute response to a standardized exercise test (identical to the pre-training test) is measured pre- and post-intervention to assess attenuation [4].

Signaling Pathways and Hormonal Regulation

The following diagram illustrates the key signaling pathways involved in the transition from acute to chronic hormonal adaptations, integrating the HPA axis, anabolic responses, and metabolic regulation.

Diagram 1: Pathway from acute hormonal responses to chronic adaptations with exercise.

The Scientist's Toolkit: Key Research Reagents and Materials

For laboratories investigating exercise endocrinology, the following tools and reagents are essential for generating high-quality data.

Table 4: Essential Research Reagents and Materials for Hormonal Analysis

Item / Solution	Function / Application	Example Use Case
EDTA or Heparin Blood Collection Tubes	Anticoagulant for plasma separation; preserves protein integrity for hormone assay.	Standard blood collection pre-, during, and post-exercise for plasma hormone analysis (e.g., catecholamines, GH). [9]
Serum Separator Tubes (SST)	Allows blood to clot for serum separation; required for many hormone immunoassays.	Collection of blood for analysis of serum cortisol, testosterone, insulin. [9]
Enzyme-Linked Immunosorbent Assay (ELISA) Kits	Quantitative detection of specific hormones using antibody-antigen binding.	Measuring concentrations of cortisol, testosterone, IL-6, and other hormones/cytokines from serum/plasma samples. [9] [11]
Radioimmunoassay (RIA) Kits	Highly sensitive quantitative method using radiolabeled antigens for hormone detection.	Historical gold standard for measuring ACTH, GH, and other peptides; used in foundational studies. [9]
Indirect Calorimetry System	Measures oxygen consumption (VOâ‚‚) and carbon dioxide production (VCOâ‚‚) to calculate energy expenditure.	Quantifying exercise intensity (%VOâ‚‚max) and substrate utilization during endurance/HIIE protocols. [5] [12]
Cycle Ergometer / Treadmill	Standardized equipment for administering endurance and HIIE exercise protocols.	Precisely controlling exercise intensity and duration for acute bouts and training interventions. [10] [5]
BTD-1	BTD-1|Benzothiadiazole Derivative|For Research	BTD-1 is a high-purity benzothiadiazole-based compound for organic electronic and photoluminescence research. For Research Use Only. Not for human or veterinary use.
Bohemine	Bohemine, CAS:16009-13-5, MF:C34H34ClFeN4O4-, MW:654.0 g/mol	Chemical Reagent

Exercise represents a potent physiological stressor that disrupts homeostasis and triggers complex neuroendocrine responses essential for adaptation. The hypothalamic-pituitary-adrenal (HPA) axis, hypothalamic-pituitary-gonadal (HPG) axis, and growth hormone-insulin-like growth factor-1 (GH-IGF-1) axis function as critical regulatory systems that integrate exercise-induced stimuli into coordinated hormonal signals [13] [5]. These systems modulate fundamental processes including energy metabolism, tissue repair, inflammatory responses, and anabolic-catabolic balance, with response patterns that vary significantly according to exercise type, intensity, duration, and individual characteristics [2]. Understanding the distinct and interactive responses of these hormonal axes provides valuable insights for optimizing athletic training, preventing overtraining syndrome, and developing targeted therapeutic interventions. This review synthesizes current evidence on the exercise-induced responses of these three key neuroendocrine systems, with particular emphasis on comparative responses across different exercise paradigms and populations.

The Hypothalamic-Pituitary-Adrenal (HPA) Axis in Exercise

Physiological Basis and Response Mechanisms

The HPA axis constitutes a primary neuroendocrine stress response system, with cascading signaling from the hypothalamus (corticotropin-releasing hormone [CRH] and arginine vasopressin [AVP]) to the pituitary (adrenocorticotropic hormone [ACTH]) and finally to the adrenal cortex (cortisol) [13] [5]. This axis regulates numerous physiological processes including metabolism, immune function, and cardiovascular activity [13]. During exercise, the HPA axis is activated primarily by neural mechanisms and metabolic challenges, particularly when exercise intensity threatens blood glucose homeostasis [5] [14]. Cortisol, the primary glucocorticoid in humans, functions to increase glucose availability via gluconeogenesis while simultaneously suppressing non-essential functions like immune and inflammatory reactions, thereby mobilizing energy reserves to meet exercise demands [13] [5].

Exercise-Type Specific HPA Axis Responses

Table 1: HPA Axis Responses to Different Exercise Types

Exercise Type	Acute Response	Chronic Adaptation	Key Influencing Factors
Endurance Exercise	Increased cortisol secretion following sufficient intensity/duration [5] [14]	Relatively increased basal cortisol levels with regular training [5]	Intensity, duration, training status, energy availability [2]
High-Intensity Interval Exercise (HIIE)	Significant cortisol increase during single bout [5]	Lower basal cortisol concentrations with regular training [5]	Work-to-rest ratio, fitness level, recovery duration [5]
Resistance Exercise	Mild HPA axis stimulation during single bout [5] [15]	Attenuated inflammatory response in elderly trainees [5]	Intensity (%1RM), volume, rest intervals [5]

The HPA axis demonstrates distinctive response patterns according to exercise modality. A single bout of endurance exercise typically stimulates cortisol increase, provided intensity and duration exceed minimum thresholds [5] [14]. The "threshold" concept posits that exercise must achieve sufficient intensity (generally >60% VO₂max) and duration (>20 minutes) to significantly activate the HPA axis [5]. High-intensity interval exercise (HIIE) generates substantial HPA axis activation during acute sessions, with regular HIIE training resulting in lowered basal cortisol concentrations—suggesting improved stress resilience [5]. Resistance exercise produces comparatively milder HPA axis stimulation, with responses dependent on training variables including intensity, volume, and rest intervals [5] [15].

Figure 1: HPA Axis Activation Pathway During Exercise. CRH = corticotropin-releasing hormone; AVP = arginine vasopressin; ACTH = adrenocorticotropic hormone. The red arrows indicate the stimulatory pathway, while blue arrows represent negative feedback mechanisms.

Experimental Protocols for HPA Axis Assessment

Standardized protocols for evaluating HPA axis response to endurance exercise typically employ incremental treadmill or cycle ergometer tests to volitional exhaustion [14]. For example, Sato and colleagues implemented a graded protocol where endurance runners exercised at low intensity for 15 minutes, moderate intensity for 15 minutes, and high intensity until exhaustion, with blood samples collected at each stage to measure cortisol dynamics [14]. HIIE protocols generally involve repeated high-intensity bouts (85-100% VOâ‚‚max) lasting 12 seconds to 4 minutes, with equal recovery intervals [5]. Resistance exercise protocols typically utilize 65-85% of one-repetition maximum (1RM) for hypertrophy-focused training or >85% 1RM for strength development, with serial hormone measurements pre-, mid-, and post-exercise [5].

The Hypothalamic-Pituitary-Gonadal (HPG) Axis in Exercise

Physiological Basis and Gender-Specific Responses

The HPG axis regulates reproductive function and sexual steroid production through coordinated secretion of hypothalamic gonadotropin-releasing hormone (GnRH), pituitary luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and end-organ hormones (testosterone and estradiol) [16]. This axis demonstrates pronounced sexual dimorphism in exercise responses. In males, acute exercise typically increases testosterone levels, while chronic training produces more variable outcomes, with evidence of suppressed testosterone in endurance athletes, particularly under conditions of low energy availability [16] [17]. In females, the HPG axis exhibits greater sensitivity to energy status, with decreased energy availability potentially inhibiting reproductive hormone secretion and causing menstrual irregularities [16].

Exercise Modality and HPG Axis Adaptation

Table 2: HPG Axis Responses in Male and Female Athletes

Parameter	Male Athletes	Female Athletes	Research Findings
Acute Exercise	Increased total and free testosterone [16] [17]	Variable testosterone and estradiol responses; menstrual cycle influences [16]	Men show more consistent acute testosterone elevations; female responses complicated by menstrual variability [16]
Chronic Training	Lower testosterone in endurance athletes; mixed responses in strength athletes [16]	Menstrual irregularities with low energy availability; relatively preserved function with adequate energy [16]	Energy availability appears to be primary determinant of HPG axis suppression in both sexes [16]
Overtraining	Suppressed testosterone, LH, and FSH; blunted response to GnRH [16] [17]	Functional hypothalamic amenorrhea; reduced bone density [16]	HPG axis suppression more readily triggered in females but occurs in both sexes with excessive training load [16]

Resistance training typically produces acute testosterone elevations in men, with responses influenced by training variables including intensity, volume, and rest intervals [17]. Long rest intervals (2-3 minutes) between heavy resistance sets promote more durable testosterone responses compared to shorter intervals [17]. Endurance training induces more variable HPG axis outcomes, with some studies demonstrating lower testosterone levels in endurance athletes compared to sedentary controls or resistance-trained athletes [16]. A well-designed randomized trial by Safarinejad and colleagues revealed that 60 weeks of high-intensity exercise (80% VO₂max) resulted in significantly lower free testosterone, FSH, and LH, with blunted responses to exogenous GnRH administration—indicating HPG axis suppression at multiple levels [16].

Methodological Considerations for HPG Axis Research

Investigating exercise-induced HPG axis changes requires careful methodological consideration. In males, testosterone assessments should account for diurnal variation, with consistent sampling times recommended [16]. In females, menstrual cycle phase significantly influences hormonal measurements, necessitating precise cycle tracking or standardization to specific phases (e.g., early follicular) for valid comparisons [16]. Low energy availability represents a major confounder in HPG axis research, particularly in "leanness sports" where weight restrictions or aesthetic demands may promote disordered eating patterns [16]. The exercise-hypogonadal male condition describes a state of reduced testosterone levels in endurance-trained males, potentially contributing to symptoms including reduced libido, erectile dysfunction, and mood disturbances [17].

Figure 2: HPG Axis Regulation and Exercise Impact. GnRH = gonadotropin-releasing hormone; LH = luteinizing hormone; FSH = follicle-stimulating hormone. Blue arrows indicate stimulatory pathways, while yellow arrows represent negative feedback mechanisms. The HPG axis demonstrates significant sexual dimorphism in exercise responses.

The Growth Hormone-Insulin-Like Growth Factor-1 (GH-IGF-1) Axis in Exercise

Exercise-Induced Activation and Anabolic Functions

The GH-IGF-1 axis plays fundamental roles in tissue growth, repair, and metabolic regulation. Exercise potently stimulates GH secretion, with circulating levels typically increasing within 15-20 minutes of exercise initiation and peaking shortly after exercise cessation [18] [19]. GH secretion patterns vary by gender, with females demonstrating earlier peak GH responses compared to males following equivalent exercise stimuli [19]. The metabolic functions of exercise-induced GH secretion include enhanced lipolysis, increased free fatty acid availability, and connective tissue stimulation [18]. While GH administration increases lean body mass in healthy adults, this effect primarily reflects expanded extracellular water content rather than functional muscle tissue accretion [18].

Training-Specific GH-IGF-1 Axis Adaptations

The GH response to exercise demonstrates intensity dependence, with high-intensity functional training incorporating rowing and resistance components producing robust GH release [18]. Interestingly, circulating IGF-1 responses to exercise show less consistency, with some studies reporting increases while others show no change or even decreases following training interventions [18] [2]. This discrepancy may reflect methodological differences in exercise protocols, assessment timing, or participant training status. Negative energy balance appears to play a major role in IGF-1 response to exercise training, potentially explaining some inconsistent findings across studies [18]. The GH-2000 project, which investigated hormonal responses to maximal exercise in elite athletes, documented coordinated increases in GH, IGF-1, IGFBP-3, and bone markers immediately post-exercise, followed by rapid return to baseline within 30-120 minutes [19].

Research Applications and Doping Control Implications

The consistent GH response to exercise has important implications for sports medicine and doping control. The GH-2000 project proposed that a combination of GH-IGF-1 axis components and bone markers could effectively detect GH doping, as these variables demonstrate differential sensitivity to exogenous GH administration versus physiological exercise [19]. Maximum exercise tests have been standardized to establish reference ranges for GH-related markers in athletic populations, accounting for factors including age, gender, and fitness level [19]. These reference ranges enable identification of aberrant hormonal patterns suggestive of pharmacological manipulation.

Figure 3: GH-IGF-1 Axis Signaling During Exercise. GH = growth hormone; IGF-1 = insulin-like growth factor-1. Blue arrows indicate stimulatory pathways, while red arrows represent both direct tissue effects and IGF-1-mediated anabolic processes.

Comparative Analysis Across Hormonal Axes

Integrated Endocrine Response to Exercise Stress

The HPA, HPG, and GH-IGF-1 axes function not in isolation but as an integrated neuroendocrine network that coordinates organismal adaptation to exercise stress. These systems demonstrate both complementary and antagonistic relationships, with cortisol exerting catabolic effects that counterbalance the anabolic functions of testosterone and GH/IGF-1 [2]. The testosterone-to-cortisol ratio has been proposed as a marker of anabolic-catabolic balance, though its utility for diagnosing overtraining syndrome remains questionable [16] [2]. Different exercise paradigms produce distinct hormonal signatures, with endurance training favoring HPA axis activation, resistance training stimulating testosterone release, and high-intensity exercise potently activating GH secretion [5] [14] [17].

Research Reagent Solutions for Exercise Endocrinology

Table 3: Essential Research Reagents for Exercise Endocrinology Studies

Reagent Category	Specific Examples	Research Applications	Technical Considerations
Hormone Assays	Salivary cortisol kits, ACTH ELISA, LC-MS/MS for steroids	Quantifying hormone concentrations in various biological matrices	Salivary vs. plasma cortisol correlations; circadian rhythm considerations [14]
Stimulation Tests	Synthetic GnRH, CRH, GHRH	Assessing functional reserve of hormonal axes	Standardized protocols required for valid comparisons [16]
Molecular Reagents	IGF-1 ELISA, IGFBP-3 RIA, P-III-P EIA	Measuring components of GH-IGF-1 axis and tissue markers	Timing critical due to rapid exercise-induced fluctuations [19]
Metabolic Assays	Lactate dehydrogenase kits, glucose oxidase reagents, NEFA kits	Correlating hormonal with metabolic responses	Enable linkage of endocrine with metabolic exercise responses [2]

Advanced reagent systems enable precise characterization of exercise-induced endocrine responses. Salivary cortisol assays provide non-invasive assessment of HPA axis activity with good correlation to plasma concentrations, though sampling and analysis protocols require standardization [14]. Molecular reagents for GH-IGF-1 axis components must account for the rapid dynamics of exercise responses, with peak concentrations typically occurring immediately post-exercise and returning to baseline within 30-120 minutes [19]. Stimulation tests using synthetic neuropeptides (GnRH, CRH, GHRH) help localize defects within hormonal axes in overtrained athletes [16].

The HPA, HPG, and GH-IGF-1 axes mediate critical adaptive responses to exercise, with distinct activation patterns according to exercise type, intensity, and individual characteristics. The HPA axis primarily responds to metabolic challenges, the HPG axis demonstrates sensitivity to energy status and training load, while the GH-IGF-1 axis shows robust activation by high-intensity exercise. Understanding these differential responses has important implications for athletic training programming, identification of overtraining syndrome, and development of exercise-based therapeutic interventions. Future research should further elucidate the molecular mechanisms governing cross-talk between these hormonal systems and explore how genetic polymorphisms influence individual response variability. Such investigations will advance personalized exercise prescription strategies targeting specific endocrine pathways for both performance enhancement and clinical applications.

Gender-Specific Basal Hormonal Landscapes and Their Influence on Exercise Responses

This guide systematically compares the basal hormonal profiles of males and females and examines their distinct physiological responses to exercise. Fundamental differences in circulating concentrations of key hormones such as testosterone, estrogen, progesterone, growth hormone (GH), and cortisol create divergent endocrine landscapes that significantly influence substrate utilization, recovery processes, and training adaptations. Experimental data from controlled studies reveal that males and females exhibit differential endocrine and metabolic responses during and following endurance exercise, resistance training, and high-intensity interval training. Understanding these gender-specific mechanisms is crucial for developing targeted therapeutic interventions, optimizing athletic training protocols, and advancing drug development for sports medicine and exercise pharmacology.

The endocrine system serves as the primary regulator of physiological responses to exercise, with significant disparities existing between genders. Following puberty, a pronounced hormonal dichotomy emerges, largely driven by differential secretion patterns of sex steroid hormones [20]. These baseline hormonal landscapes establish fundamentally different physiological environments that shape how males and females respond to and adapt to various exercise stimuli.

Circulating testosterone concentrations represent the most striking gender-divergent hormone, with men exhibiting levels 15 to 20-fold higher than women post-puberty [20]. This substantial variance creates a powerful anabolic environment in males that profoundly influences muscle mass, strength, and hemoglobin levels. Conversely, females experience cyclical fluctuations of estrogen and progesterone throughout the menstrual cycle, creating a more dynamic hormonal environment that modulates exercise metabolism and recovery [21]. These basal differences provide the foundation for gender-specific exercise responses observed across multiple physiological domains.

Basal Hormonal Profiles: A Comparative Analysis

Circulating Sex Steroids

The most profound differences in basal hormonal landscapes concern the sex steroids, which establish fundamentally different anabolic environments and metabolic regulation systems between genders.

Table 1: Gender-Specific Basal Hormonal Profiles

Hormone	Male Concentration	Female Concentration	Fold Difference
Testosterone	290-1000 ng·dL⁻¹ (10-35 nM) [22]	14-65 ng·dL⁻¹ (0.5-2.5 nM) [22]	15-20x higher in males [20]
Estrogen	Low, stable	Cyclical: 200-300 pg·mL⁻¹ at peak [23]	Substantially higher in females
Progesterone	Low, stable	Cyclical: 8-10 ng·mL⁻¹ at peak [23]	Substantially higher in females

Males maintain relatively stable sex hormone levels, while females experience significant cyclical variations throughout the menstrual cycle phases [21]. The menstrual cycle is characterized by extraordinary variation between individuals, with estrogen peaking at approximately 200-300 pg·mL⁻¹ around day 12 (during the follicular phase), while progesterone reaches 8-10 ng·mL⁻¹ at approximately day 20 (during the luteal phase) [23]. These cyclical fluctuations create a constantly changing hormonal environment that influences various aspects of exercise physiology.

Metabolic and Stress Hormones

Beyond sex steroids, other hormones involved in metabolism and stress response also demonstrate gender-specific patterns, though often with less dramatic differences than those observed with sex steroids.

Table 2: Metabolic and Stress Hormone Profiles

Hormone	Male Characteristics	Female Characteristics	Response to Exercise
Growth Hormone (GH)	Lower acute response	Higher acute exercise response [2]	Attenuated with training in both
Cortisol	Higher response [2]	Attenuated response [2]	Increased during intense exercise
IGF-1	Higher baseline [2]	Lower baseline [2]	Modest increases with training

These baseline differences establish distinct anabolic-catabolic environments that influence how each gender responds to training stimuli. The catabolic hormone cortisol shows a more pronounced response in males during exercise, while females exhibit a greater acute GH response [2].

Experimental Approaches to Studying Gender-Specific Exercise Responses

Endurance Exercise Protocol

Objective: To characterize gender differences in substrate utilization and endocrine responses during recovery from endurance exercise.

Methodology: A controlled study compared trained male (n=6) and female (n=6) endurance runners following a 75-minute run at 70% VOâ‚‚peak [24]. Key methodological elements included:

• Dietary Control: Participants consumed a standardized euenergetic diet (1.8 g·kg⁻¹·d⁻¹ protein, 26% fat, 58% carbohydrate) for 8 days prior to testing [24]
• Menstrual Cycle Control: Female participants were tested during the early follicular phase (days 1-7) when estrogen and progesterone are lowest [24]
• Fitness Matching: Subjects were matched based on training volume (≥56 km·wk⁻¹) and fitness level (VOâ‚‚peak values) [24]
• Blood Sampling: Collected at rest and during 3.5-hour recovery period for glucose, lactate, free fatty acids (FFAs), insulin, cortisol, GH, and free IGF-1 [24]

Key Findings: During the recovery period, females experienced significant increases in glucose, lactate, and insulin (p<0.05), while no changes were noted in males. Conversely, males demonstrated increases in GH and decreases in IGF-I levels (p<0.05), with no changes observed in females. FFA levels increased during recovery in both genders without significant between-group differences [24].

Resistance Training Protocol

Objective: To investigate associations between acute exercise-induced hormone responses and resistance training adaptations.

Methodology: A 12-week resistance training study with 56 young men examined correlations between acute hormonal responses and training adaptations [25]:

• Training Program: 5 days per week training using both upper- and lower-body exercises
• Blood Analysis: Serum growth hormone, free testosterone, IGF-1, and cortisol measured at rest and at 0, 30, 60, 90, and 120 minutes post-exercise at week 7
• Outcome Measures: Lean body mass (via DXA), muscle fibre cross-sectional area (histochemical planimetry), and leg press strength

Key Findings: No significant correlations were found between exercise-induced elevations of GH, free testosterone, and IGF-1 with gains in lean body mass or strength. However, cortisol responses correlated with changes in lean body mass (r=0.29, p<0.05) and type II fibre cross-sectional area (r=0.35, p<0.01) [25].

High-Intensity Interval Training (HIIT) vs. Traditional Resistance Training (TRT)

Objective: To compare the effects of HIIT and TRT on hormonal profiles in young women.

Methodology: A 10-week intervention study with 72 young women randomly assigned to HIIT or TRT groups [26]:

• Training Protocols: Both groups trained 3 times per week for 10 weeks
• HIIT Protocol: Alternating 2-minute brisk walking with 2-minute easy walking, progressively increasing to 50 minutes daily at 75-90% maximum heart rate
• TRT Protocol: Elastic bands, light weights, and bodyweight exercises targeting major muscle groups
• Hormonal Assessment: Estrogen, testosterone, FSH, prolactin, and LH measured pre- and post-intervention

Key Findings: Both interventions significantly modulated hormonal profiles. The HIIT group showed a 150% increase in estrogen versus 72.3% in the TRT group. Testosterone decreased by 58% in the HIIT group versus 49% in the TRT group. Both groups showed modest decreases in FSH (HIIT: 6%; TRT: 7.7%) and prolactin (HIIT: 5%; TRT: 2.1%), with no significant changes in LH [26].

Signaling Pathways in Hormonal Exercise Responses

The endocrine response to exercise involves complex interactions between multiple hormonal axes. The following diagram illustrates the primary signaling pathways activated during physical exertion:

Figure 1: Gender-Specific Hormonal Signaling Pathways in Exercise Response

The hormonal exercise response model (HERM) illustrates how exercise triggers rapid sympathetic nervous system activation, releasing catecholamines and altering insulin and glucagon levels [2]. As exercise continues, the hypothalamus stimulates the pituitary gland, which releases hormones like cortisol. The model demonstrates how these responses evolve from neural to feedback-driven mechanisms as exercise duration increases, with significant gender-based divergences in the HPG axis modulation [2].

The Scientist's Toolkit: Essential Research Reagents

Investigating gender-specific hormonal responses to exercise requires specialized reagents and methodologies to ensure accurate hormone quantification and proper experimental control.

Table 3: Essential Research Reagents and Methodologies

Reagent/Methodology	Application	Technical Considerations
LC-MS (Liquid Chromatography-Mass Spectrometry)	Gold-standard for testosterone quantification [20]	Essential for accurate measurement of low female testosterone levels
Immunoassays (Immulite system)	GH, free testosterone, IGF-1, cortisol measurement [25]	Solid-phase, two-site chemiluminescence immunometric assays
DXA (Dual-energy X-ray Absorptiometry)	Lean body mass assessment [25]	Coefficient of variation <2% for repeated scans; different methodologies may be used between genders
Hydrostatic Weighing vs. DXA	Body composition determination	Males: hydrostatic weighing; Females: DXA - methodologies must be reported [24]
Menstrual Cycle Tracking	Standardizing female testing phases	Confirm phase with plasma estradiol measurements; early follicular phase (days 1-7) recommended [24]
Dietary Standardization	Controlling for nutritional confounders	Euenergetic diets with fixed macronutrient ratios (e.g., 1.8 g·kg⁻¹·d⁻¹ protein) for 8+ days pre-testing [24]
ML303	ML303, MF:C21H16F3N3O2, MW:399.4 g/mol	Chemical Reagent
IMT1B	IMT1B|POLRMT Inhibitor|For Research Use	IMT1B is a potent, selective POLRMT inhibitor that targets mitochondrial transcription for cancer research. For Research Use Only. Not for human use.

The gender-specific basal hormonal landscapes create fundamentally different physiological environments that significantly influence exercise responses and adaptations. The experimental evidence demonstrates that females experience different substrate utilization patterns during recovery from endurance exercise, characterized by increased glucose, lactate, and insulin responses compared to males [24]. Resistance training adaptations show complex relationships with acute hormonal responses, with cortisol demonstrating unexpected positive correlations with lean mass gains in males [25]. Exercise interventions like HIIT and TRT differentially modulate hormonal profiles in women, with HIIT producing more pronounced effects on estrogen elevation [26].

These findings have significant implications for drug development and therapeutic interventions targeting exercise performance, recovery, and body composition. Pharmaceutical approaches should account for the profoundly different hormonal environments between genders, particularly the 15-20 fold difference in testosterone concentrations [20] and the cyclical variations in estrogen and progesterone in females [21]. Future research should employ gold-standard methodologies for hormone assessment and menstrual cycle verification to advance our understanding of how exercise prescriptions can be optimized for each gender across the lifespan.

Modulation of Reproductive Hormones (Estrogen, Testosterone, FSH, LH) by Exercise Modality

The modulation of reproductive hormones by physical activity is a critical area of investigation within exercise endocrinology, with significant implications for metabolic health, reproductive function, and performance optimization across diverse populations. Understanding how different exercise modalities distinctly influence the hypothalamic-pituitary-gonadal (HPG) axis provides a scientific foundation for developing targeted, evidence-based interventions. This guide objectively compares the hormonal responses elicited by predominant exercise modalities—high-intensity interval training (HIIT) and traditional resistance training (TRT)—by synthesizing findings from key controlled interventions. It is structured to serve researchers, scientists, and drug development professionals engaged in comparative studies of hormonal responses to exercise, presenting detailed experimental protocols, quantitative outcomes, and essential research tools.

Experimental Protocols and Methodologies

Key Comparative Intervention Study

A foundational 10-week randomized controlled trial (RCT) directly compared the effects of HIIT and TRT on reproductive hormones in young women [27] [28].

• Participants: 72 healthy, physically active female college students were recruited and randomly assigned to either a HIIT group (n=36) or a TRT group (n=36). The study employed an RCT design to minimize selection bias [27].
• Exclusion Criteria: Screening excluded individuals based on pregnancy, smoking, Type II diabetes, acute or chronic cardiovascular disorders, abnormal menstrual function (e.g., amenorrhea or irregular menstruation), contraindications to physical activity, and the use of hormonal supplementation [27].
• Study Design and Duration: The intervention spanned 10 weeks, with training sessions scheduled three times per week, culminating in 30 total sessions. A mandatory 24-hour recovery period was enforced between sessions. Pre- and post-intervention performance and hormonal assessments were conducted [27].
• HIIT Protocol: Participants began with a 20-minute protocol of alternating 2-minute bouts of brisk walking and easy walking. The total exercise duration was progressively increased by 5 minutes weekly, eventually reaching 50 minutes of daily exercise. The intensity was maintained at 75–90% of maximum heart rate, monitored using Polar watches [27].
• TRT Protocol: The TRT group performed exercises targeting major muscle groups twice weekly for approximately 30 minutes per session. The regimen utilized elastic bands, light weights, and adapted bodyweight exercises. Intensity was set at 60–80% of one-repetition maximum (1RM), progressively increased as tolerated [27].
• Hormonal Assessment: Blood samples were collected after a minimum 4-hour fast, both before and after the 10-week intervention. Samples were frozen, and assays were conducted using standardized methodologies in a single batch at the study's conclusion to minimize inter-assay variability [27].

Supporting Experimental Evidence

Other studies provide complementary methodological insights and findings:

• Integrated Exercise in Eumenorrheic Women: A 16-week, single-blinded RCT investigated the effects of an integrated exercise plan performed three times per week on total testosterone levels. Testosterone was measured pre-, mid-, and post-intervention across the follicular, mid-cycle, and luteal phases of the menstrual cycle, demonstrating the importance of phase-specific analysis [29].
• Exercise in Postmenopausal Women: A review of RCTs assessing the impact of physical activity on androgens in postmenopausal women found that the type of exercise significantly alters hormonal outcomes. For instance, aerobic exercise tended to decrease total testosterone, whereas resistance training increased it [30].
• Factors Modulating Hormonal Response: A comprehensive review highlights that the hormonal response to exercise is not uniform but is modulated by factors including exercise type (endurance vs. resistance), intensity, duration, resting periods, and participant characteristics (e.g., age, training status, and body composition) [31].

Quantitative Hormonal Outcomes

The 10-week comparative intervention yielded significant, modality-dependent changes in key reproductive hormones, summarized in the table below.

Table 1: Comparative Effects of a 10-Week HIIT vs. TRT Intervention on Hormonal Profiles in Young Women [27] [28]

Hormone	HIIT Change (Pre- to Post-Intervention)	TRT Change (Pre- to Post-Intervention)	Notes
Estrogen	+150%	+72.3%	Both interventions produced significant increases, with HIIT inducing a markedly greater response.
Testosterone	-58%	-49%	Both interventions produced significant decreases.
Follicle-Stimulating Hormone (FSH)	-6%	-7.7%	Both interventions produced small but significant decreases.
Prolactin (PL)	-5%	-2.1%	Both interventions produced small but significant decreases.
Luteinizing Hormone (LH)	No Significant Change	No Significant Change	Levels remained stable in both groups.

Interpretation of Hormonal Data

• Estrogen and Testosterone: The dramatic increase in estrogen, particularly following HIIT, suggests a potent stimulus for aromatization or ovarian production. The concurrent decrease in testosterone may indicate an increased conversion rate to estrogen or a differential regulation of the HPG axis [27].
• FSH and Prolactin: The modest reductions in FSH and prolactin point to an exercise-induced modulation of pituitary secretion, potentially reflecting an improved energy balance and reduced metabolic stress following consistent training [27].
• LH Stability: The lack of significant change in LH levels suggests that the fundamental pulsatile release of this hormone from the pituitary may be less affected by these specific exercise modalities over the long term compared to other hormones [27].

Signaling Pathways and Mechanistic Insights

The neuroendocrine response to exercise involves complex interactions along the HPG axis. The following diagram synthesizes the primary signaling pathways modulated by different exercise modalities, as evidenced by the experimental data.

Hormonal Exercise Response Pathway

Diagram 1: Exercise Modality Modulation of the HPG Axis. This pathway illustrates how HIIT and TRT influence reproductive hormone secretion via the brain-pituitary-gonad feedback loop, leading to the distinct hormonal outcomes quantified in Table 1.

The Scientist's Toolkit: Research Reagent Solutions

The following table details essential materials and methodologies used in the featured experiments, providing a reference for replicating and validating these findings.

Table 2: Key Research Reagents and Methodologies for Hormonal Exercise Studies

Item / Methodology	Specific Example / Function	Research Application
Participant Screening	Predefined exclusion criteria (e.g., hormonal contraception, menstrual dysfunction) [27].	Ensures a homogeneous sample, controlling for confounding variables in endocrine assessments.
Exercise Intensity Monitoring	Polar heart rate watches [27].	Objectively quantifies and ensures adherence to prescribed exercise intensity (e.g., 75-90% max HR for HIIT).
Strength Assessment	One-Repetition Maximum (1RM) testing [27].	Determines initial strength levels and sets precise training loads for TRT (e.g., 60-80% of 1RM).
Hormonal Assay	Standardized batch analysis of blood samples [27].	Minimizes technical variance; ensures reliability and comparability of pre- and post-intervention hormone levels (Estrogen, Testosterone, FSH, LH, Prolactin).
Menstrual Cycle Phase Assessment	Hormonal measurement across follicular, ovulatory, and luteal phases [29].	Controls for and investigates the confounding effects of natural hormonal fluctuations in eumenorrheic women.
Data Collection Tool	Daily exercise logs with Ratings of Perceived Exertion (RPE) [27].	Provides subjective and objective data on training adherence, intensity, and physiological response.
VDM11	VDM11 Anandamide Uptake Inhibitor|Research Compound	VDM11 is a potent anandamide transport inhibitor for researching neuroinflammation, reward-seeking behavior, and cough reflex. For Research Use Only. Not for human or veterinary use.
SIM1	SIM1 Antibody for Research

This guide synthesizes experimental evidence demonstrating that exercise modality is a decisive factor in modulating reproductive hormone profiles. The data unequivocally show that while both HIIT and TRT are potent endocrine stimuli, they elicit distinct response patterns—most notably, HIIT induces a substantially greater increase in estrogen levels compared to TRT. The detailed methodologies and reagent solutions provided establish a framework for reproducing these findings and extending this research. For the scientific and drug development communities, these insights are invaluable. They underscore the potential of tailoring exercise prescriptions to achieve specific hormonal outcomes, thereby informing the development of non-pharmacological therapeutic strategies and providing a comparative physiological basis for evaluating hormonal interventions across diverse populations. Future research should continue to elucidate the molecular mechanisms underlying these modality-specific effects and explore their long-term implications for health and disease.

Research Methodologies for Capturing Hormonal Dynamics in Exercise Physiology

The precise measurement of hormonal output is critical for research in exercise physiology, sports science, and drug development. However, the standardization of exercise protocols remains a significant challenge, as variations in intensity, volume, and modality produce markedly different endocrine responses [2]. Understanding these nuances is essential for researchers designing clinical trials, developing therapeutic exercise interventions, and evaluating the efficacy of pharmacological agents targeting metabolic and endocrine pathways.

Hormonal responses to exercise are governed by a complex interplay of factors. The Hormonal Exercise Response Model (HERM) describes these responses in three phases: initial rapid sympathetic nervous system activation, subsequent hypothalamic-pituitary stimulation, and finally, involvement of additional hormones from peripheral glands during prolonged activity [2]. This systematic review synthesizes experimental data from controlled studies to compare how key exercise variables—intensity, volume, and modality—impact hormonal output across different populations, providing a framework for protocol standardization in research settings.

Hormonal Responses to Exercise: Key Axes and Mechanisms

Major Endocrine Axes Activated by Exercise

Physical activity simultaneously engages multiple hormonal systems that regulate metabolism, fluid balance, and tissue adaptation [2]. The hypothalamic-pituitary-adrenal (HPA) axis and hypothalamic-pituitary-gonadal (HPG) axis are particularly responsive to exercise stressors, with output dependent on both acute exercise stimuli and chronic training status.

The table below summarizes the primary hormonal systems involved in exercise responses:

Table 1: Major Hormonal Axes Activated by Exercise

Hormonal Axis	Key Hormones	Primary Exercise-Related Functions	Response Patterns
Hypothalamic-Pituitary-Adrenal (HPA)	Cortisol, ACTH	Metabolic fuel mobilization, stress response, inflammatory modulation	Increases with intensity/duration; attenuated after training [2]
Hypothalamic-Pituitary-Gonadal (HPG)	Testosterone, Estradiol, LH, FSH	Anabolic processes, tissue repair, body composition regulation	Variable by sex/intensity; suppressed with low energy availability [2]
Growth Hormone (GH) Axis	GH, IGF-1	Protein synthesis, muscle hypertrophy, metabolic regulation	Potently stimulated by exercise; resistance exercise triggers greater response [2]
Adrenergic System	Epinephrine, Norepinephrine	Cardiovascular function, substrate mobilization, metabolic rate	Rapid increase with exercise onset; intensity-dependent [2]
Metabolic Hormones	Insulin, Glucagon	Glucose homeostasis, nutrient storage and utilization	Insulin decreases during activity; interacts with exercise timing/nutrition [2]

Visualization of Exercise-Induced Hormonal Signaling Pathways

The following diagram illustrates the primary hormonal pathways activated during physical exercise and their interrelationships:

Figure 1: Exercise-Induced Hormonal Signaling Pathways. This diagram illustrates the primary endocrine pathways activated during physical activity, highlighting the complex interplay between different glands, hormones, and their effects. The HPA axis (red), anabolic pathways (blue), and metabolic regulators (green) respond differentially based on exercise variables.

Comparative Analysis of Exercise Protocols and Hormonal Outcomes

Intensity-Dependent Hormonal Responses

Exercise intensity serves as a primary determinant of hormonal output, with different thresholds eliciting distinct endocrine profiles. Research has classified exercise intensity into three primary categories with characteristic hormonal signatures:

Table 2: Intensity-Dependent Hormonal Responses to Exercise

Intensity Classification	Definition	Cortisol Response	Testosterone Response	Growth Hormone Response	Key Research Findings
Low-Moderate Intensity Continuous	Below second ventilatory threshold, <4 mmol/L blood lactate, <87% HRmax [32]	Moderate increase	Minimal change	Moderate increase	23% increase in mitochondrial content; optimal for capillarization [32]
High-Intensity Interval Training (HIIT)	>87% HRmax or VOâ‚‚ max, above second ventilatory threshold [32]	Significant increase	Significant increase (+28%) [33]	Substantial increase	27% increase in mitochondrial content; most time-efficient for VOâ‚‚ max gains [32]
Sprint Interval Training (SIT)	Maximal or supramaximal efforts (4-90 seconds) [32]	Pronounced increase	Variable; depends on recovery	Extreme increase	27% mitochondrial biogenesis; 2-3x more efficient per time than endurance training [32]

The data reveals a clear intensity-response relationship for catabolic and anabolic hormones. HIIT protocols produce particularly potent endocrine responses, with one study showing 28% increases in testosterone and 16-30% increases in free testosterone in previously inactive middle-aged adults [33]. Concurrently, HIIT reduced cortisol levels by 10-23%, suggesting an improved anabolic-catabolic balance [33].

Volume-Modulated Hormonal Adaptations

Training volume, typically quantified as total work performed, interacts with intensity to modulate hormonal output. Comparative studies have examined how volume affects hormonal responses:

Table 3: Volume-Modulated Hormonal Adaptations Across Exercise Modalities

Training Protocol	Volume Parameters	Testosterone Response	Cortisol Response	Growth Hormone Response	Key Findings
High-Intensity Training (HIT)	1 set to momentary muscular failure + drop-sets [34]	Significant increases	Not reported	Not reported	Significantly greater muscular performance gains vs. higher volume in 8 of 9 exercises [34]
Bodybuilding (3ST)	3 sets to self-determined repetition maximum [34]	Moderate increases	Not reported	Not reported	Lower effect sizes for strength gains compared to HIT despite higher volume [34]
Blood Flow Restriction (BFR)	30-15-15-15 reps at 30% 1RM [35]	No significant change	No significant change	423% increase with active recovery [35]	Active recovery between BFR sets significantly enhanced GH response vs. passive recovery [35]

Volume appears to interact with intensity in determining hormonal responses. Interestingly, low-volume high-intensity training often produces superior hormonal and performance adaptations compared to higher-volume protocols, suggesting that intensity may outweigh volume in stimulating anabolic endocrine responses [34]. The implementation of advanced techniques such as blood flow restriction further modulates this relationship, allowing substantial hormonal responses with minimal external load [35].

Modality-Specific Hormonal Signatures

Exercise modality distinctly shapes hormonal output through differences in muscle fiber recruitment patterns, metabolic demands, and physiological stress:

Table 4: Modality-Specific Hormonal Responses in Comparative Studies

Exercise Modality	Protocol Details	DHEAS Response	Cortisol Response	Testosterone Response	Notable Population Effects
Concurrent Training (PAR)	150 min/week at 60-65% HRR + resistance training [33]	+14%	-17%	No significant change	Lower steroidogenic response despite higher volume [33]
HIIT	40-65 min/week at >95% VOâ‚‚ max [33]	+14%	-10%	+28%	Superior anabolic response despite lower time commitment [33]
HIIT + EMS	HIIT with whole-body electromyostimulation [33]	+20%	-23%	+16%	Enhanced steroidogenic response; combined stimulus most potent [33]
Very Low Volume HIIT	<30 min/week at ≥80% HRmax [36]	Not reported	Not reported	Not reported	Improved VO₂max (+3.1 mL/kg/min) and metabolic syndrome severity [36]

Modality comparisons reveal that high-intensity interval training consistently produces robust endocrine responses even at very low volumes (<30 minutes per week) [36]. The addition of whole-body electromyostimulation to HIIT further enhances steroidogenic responses, particularly for DHEAS (+20%) [33], suggesting synergistic effects when combining modalities.

Experimental Protocols and Methodologies

Detailed Methodologies from Key Studies

HIIT vs. Moderate-Intensity Training in Middle-Aged Adults

Study Design: 12-week randomized controlled trial with parallel-group design [33].

Participants: 67 (36 women) physically inactive middle-aged adults (45-65 years).

Intervention Groups:

• PAR Group: Concurrent training based on international physical activity recommendations (150 min/week at 60-65% HRR for aerobic training + ~60 min/week resistance training at 40-50% 1RM)
• HIIT Group: High-intensity interval training (40-65 min/week at >95% VOâ‚‚ max in long intervals and >120% VOâ‚‚ max in short intervals)
• HIIT+EMS Group: HIIT plus whole-body electromyostimulation

Measurements: Plasma steroid hormone levels (DHEAS, cortisol, testosterone, free testosterone, SHBG) assessed pre- and post-intervention.

Key Findings: HIIT and HIIT+EMS produced significant increases in testosterone (+28% and +16%) and free testosterone (+30% and +18%), while all exercise groups showed increased DHEAS and reduced cortisol [33].

Low Volume HIT vs. Bodybuilding Training

Study Design: 10-week randomized trial with two experimental groups [34].

Participants: 30 participants (13 males, 17 females) who were healthy university sports students.

Intervention Groups:

• HIT Group: Single set of each exercise to momentary muscular failure plus drop-sets
• 3ST Group: Three sets of each exercise to self-determined repetition maximum

Training Frequency: 2 sessions/week with at least 48 hours between sessions.

Exercises: Chest press, heel raise, rear deltoid, elbow flexion, seated row, knee extension, knee flexion, abdominal flexion, push-ups in circuit fashion.

Measurements: Muscular performance (10RM testing), body composition (bioelectrical impedance), subjective assessments.

Key Findings: HIT group demonstrated significantly greater muscular performance gains for 3 of 9 tested exercises and larger effect sizes for 8 of 9 exercises despite substantially lower volume [34].

Research Reagent Solutions and Essential Materials

The following table details key reagents and materials essential for conducting hormonal response research in exercise physiology:

Table 5: Essential Research Reagents and Materials for Exercise Endocrinology Studies

Reagent/Material	Specific Application	Function/Measurement Purpose	Example from Studies
Enzyme Immunoassay Kits	Hormone quantification in blood, saliva	Measure cortisol, testosterone, GH, IGF-1 levels	Pre- and post-intervention steroid hormone measurement [33]
Blood Collection Equipment	Serum/plasma sampling	Obtain samples for hormonal analysis	Overnight-fasted blood samples pre-/post-intervention [36]
Metabolic Analyzers	VOâ‚‚ max testing, lactate threshold	Assess cardiopulmonary fitness, determine intensity zones	Maximal oxygen uptake assessment in obese MetS patients [36]
Bioelectrical Impedance Devices	Body composition analysis	Estimate muscle mass, fat mass, total body water	Tanita MC-180 for body composition tracking [34]
Heart Rate Monitoring Systems	Exercise intensity regulation	Ensure target intensity zones are maintained	Training at >95% VOâ‚‚ max for HIIT protocols [33]
Blood Flow Restriction Cuffs	BFR training implementation	Create ischemic conditions for low-load training	Pneumatic cuffs at 60% arterial occlusion pressure [35]
Resistance Training Equipment	Standardized exercise protocols	Ensure consistent training stimuli across participants	Nautilus resistance machines for controlled training [34]

Discussion and Research Implications

Standardization Challenges and Considerations

The evidence demonstrates substantial heterogeneity in hormonal responses to exercise, complicating protocol standardization. Several key factors contribute to this variability:

Individual Response Determinants: Hormonal responses to standardized exercise protocols show considerable inter-individual variation influenced by genetics, age, sex, biological rhythms, nutritional status, training history, and physiological characteristics [2] [37]. This variability underscores the need for personalized exercise prescription in research settings.

Baseline Fitness Status: The magnitude of hormonal adaptation is inversely related to baseline fitness, with untrained individuals showing more pronounced responses [32]. Well-trained participants (VO₂ max ~62.2 mL·kg⁻¹·min⁻¹) demonstrate attenuated responses compared to untrained (VO₂ max ~34.8 mL·kg⁻¹·min⁻¹) or moderately trained individuals (VO₂ max ~48.8 mL·kg⁻¹·min⁻¹) [32].

Temporal Patterns: Hormonal responses evolve throughout exercise duration, transitioning from neural-driven to feedback-regulated mechanisms [2]. The timing of biological sample collection relative to exercise sessions therefore critically impacts measured hormonal concentrations.

Methodological Recommendations for Research

Based on the synthesized evidence, the following recommendations can enhance protocol standardization:

• Intensity Prescription: Utilize objective measures (%VOâ‚‚ max, %HRmax, lactate thresholds) rather than relative perceived exertion for intensity standardization [32].

• Volume Considerations: Recognize that low-volume high-intensity protocols often produce robust endocrine responses, potentially offering superior efficiency for certain research applications [34] [36].

• Modality Selection: Carefully match exercise modality to research questions, considering that combined training approaches (e.g., HIIT+EMS) may produce synergistic effects [33].

• Participant Stratification: Account for baseline fitness, sex, age, and training history in study design and analysis, as these factors significantly moderate hormonal responses [2] [32].

The following diagram illustrates a standardized approach to exercise protocol design for hormonal research:

Figure 2: Exercise Protocol Decision Framework for Hormonal Research. This diagram provides a systematic approach to selecting exercise intensity, volume, and modality based on research questions and participant characteristics, with evidence-based recommendations for specific applications.

This analysis demonstrates that exercise intensity, volume, and modality systematically influence hormonal output through distinct physiological pathways. High-intensity protocols consistently produce robust anabolic hormonal responses, even at very low volumes, while moderate-intensity endurance training offers distinct benefits for metabolic and capillary adaptations. The interaction between these variables underscores the need for precise protocol standardization in research settings.

Future studies should prioritize individualized exercise prescription that accounts for baseline fitness, sex, age, and training history to reduce response heterogeneity. Additionally, research exploring the molecular transducers of exercise—genomic, proteomic, transcriptomic, and metabolomic factors—will further elucidate the mechanisms underlying hormonal response variation [37]. Such advances will enhance the precision of exercise protocols in research applications, ultimately strengthening the evidence base for both exercise and pharmacological interventions targeting endocrine pathways.

Blood biomarker analysis has become a cornerstone of modern biomedical research, providing critical insights into physiological status, disease mechanisms, and intervention efficacy. Within exercise science, the accurate measurement of hormonal biomarkers is particularly crucial for understanding the complex endocrine responses to physical activity across different populations. The reliability of these measurements, however, is profoundly influenced by pre-analytical variables ranging from sample collection methods to analytical technique selection. This comprehensive guide examines best practices in blood biomarker analysis, focusing specifically on applications in exercise endocrinology research. By comparing standardized protocols and advanced assay technologies, we provide researchers with evidence-based methodologies to enhance data quality, improve cross-study comparability, and advance our understanding of hormonal dynamics in response to exercise.

Standardized Procedures for Blood Sampling and Processing

Critical Pre-Analytical Variables in Blood Collection

The integrity of biomarker analysis begins at the moment of sample collection, where numerous pre-analytical factors can significantly influence results. For hormonal biomarkers commonly studied in exercise research, including testosterone, estrogen, cortisol, and growth hormone, standardized procedures are essential for obtaining reliable data [38].

Key sampling considerations include the time of day for blood collection, participant fasting status, needle characteristics, and collection tube type. Research indicates that morning sampling is generally preferred for hormonal assays due to diurnal variations in many hormones [38]. Fasting status should be standardized and documented, as nutrient intake can influence certain hormonal levels. For venipuncture, 21-gauge needles (with a range of 19-24 gauge) are recommended, using gentle drawing techniques to prevent hemolysis that can compromise sample quality [38].

Collection tube selection represents another critical decision point. Ethylenediaminetetraacetic acid (EDTA) plasma tubes are generally recommended for most biomarker analyses, though researchers should confirm compatibility with specific target biomarkers [38]. Comparative studies have shown that biomarker levels can vary significantly based on tube additives, with samples collected in sodium citrate typically showing lower levels and lithium heparin samples showing higher levels for certain biomarkers compared to EDTA plasma [38].

Sample Processing and Storage Protocols

Post-collection processing parameters significantly impact biomarker stability and measurement accuracy. The time from collection to centrifugation should be minimized, with processing ideally occurring within 1 hour for biomarkers sensitive to degradation, such as total tau [38]. For more stable biomarkers including neurofilament light chain (NfL), glial fibrillary acidic protein (GFAP), and phosphorylated tau (p-tau181), samples remain stable for up to 24 hours at room temperature [38].

Centrifugation parameters should be standardized at 10 minutes at 1,800 × g at either room temperature or 4°C [38]. Following centrifugation, samples should be aliquoted and frozen at -80°C as soon as possible. If immediate freezing is not feasible, samples can be stored at 2°C-8°C for up to 24 hours or at -20°C for 2-14 days [38]. Freeze-thaw cycles should be minimized, with recommendations limiting cycles to two or fewer for most biomarkers [38].

Table 1: Standardized Protocols for Blood Sample Processing and Storage

Processing Stage	Recommendation	Notes
Time to Centrifugation	Within 1 hour	Critical for unstable biomarkers (e.g., t-tau); up to 3-24 hours for more stable biomarkers
Centrifugation Parameters	10 min at 1,800 × g, RT or 4°C	Standardized force and time critical
Time to Freezing	As soon as possible after centrifugation	Temporary storage at 2°C-8°C (<24 hours) acceptable
Long-term Storage	-80°C	Consistent ultra-low temperature essential
Freeze-Thaw Cycles	≤ 2 cycles	Document exact numbers if exceeding one cycle
Aliquot Volume	250-1,000 μL in polypropylene tubes	Fill tubes to at least 75% capacity to minimize oxidation

Sample Processing Workflow

The following diagram illustrates the complete standardized workflow for blood sample processing from collection to storage, integrating the critical control points discussed above:

Diagram 1: Blood Sample Processing Workflow. This diagram outlines the critical steps and control points in standardized blood sample processing, highlighting time-sensitive procedures and quality control measures essential for maintaining biomarker integrity.

Analytical Techniques: Comparing Immunoassay and Mass Spectrometry

Methodological Comparison for Hormone Analysis

The selection of analytical methodology represents a fundamental decision in hormone biomarker research, with immunoassays and mass spectrometry emerging as the two primary technologies. Each approach offers distinct advantages and limitations that researchers must consider based on their specific analytical requirements.

Enzyme-linked immunosorbent assay (ELISA) techniques have been widely used for hormonal biomarker quantification due to their relatively low cost, technical accessibility, and high-throughput capabilities. However, recent comparative studies have revealed significant limitations in ELISA performance, particularly for salivary sex hormone analysis. A 2025 comparative study demonstrated poor performance of ELISA for measuring salivary estradiol and progesterone, with testosterone showing somewhat better but still suboptimal validity compared to mass spectrometry [39].

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a superior analytical technique for hormonal biomarker quantification, offering enhanced specificity, sensitivity, and accuracy. Despite its technical challenges and higher operational costs, LC-MS/MS provides more reliable quantification of steroid hormones including estradiol, progesterone, and testosterone [39]. The technology's capacity to distinguish between structurally similar molecules reduces cross-reactivity issues common in immunoassays, making it particularly valuable for measuring low-concentration hormones in complex matrices.

Assay Selection Decision Framework

The following diagram outlines a systematic approach for selecting appropriate analytical methods based on research objectives, technical resources, and required performance characteristics:

Diagram 2: Assay Selection Decision Framework. This diagram provides a structured approach for selecting appropriate analytical methodologies based on research requirements, technical resources, and performance considerations, highlighting the superior validity of LC-MS/MS for steroid hormone analysis.

Table 2: Comparative Analysis of Immunoassay and Mass Spectrometry Techniques

Parameter	Immunoassay (ELISA)	Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)
Analytical Principle	Antibody-antigen binding with enzymatic detection	Mass-to-charge ratio separation and detection
Specificity	Moderate (cross-reactivity concerns)	High (physical separation reduces interference)
Sensitivity	Variable; matrix-dependent	Consistently high, even in complex matrices
Multiplexing Capacity	Limited without specialized panels	Broad; capable of analyzing multiple analytes
Throughput	High	Moderate to high (improving with automation)
Technical Expertise	Moderate	Advanced expertise required
Equipment Costs	Lower	Significantly higher
Validation Data	Poor performance for salivary estradiol and progesterone [39]	Superior accuracy for sex steroid quantification [39]

Exercise Endocrinology: Methodological Considerations

Hormonal Responses to Exercise Interventions

Research examining hormonal responses to exercise requires careful consideration of both sampling protocols and exercise intervention design. Studies have demonstrated that both high-intensity interval training (HIIT) and traditional resistance training (TRT) significantly modulate hormonal profiles in women [27]. A 10-week intervention study with 72 young women showed that both training modalities induced significant increases in estrogen (HIIT: 150%; TRT: 72.3%) and decreases in testosterone (HIIT: 58%; TRT: 49%), FSH (HIIT: 6%; TRT: 7.7%), and prolactin (HIIT: 5%; TRT: 2.1%) [27].

The timing of blood sampling relative to exercise sessions is particularly important for capturing acute hormonal responses. Research on integrated exercise approaches in eumenorrheic women has demonstrated that testosterone levels increase immediately after exercise but decrease below pre-exercise levels within 24 hours [29]. This pattern emphasizes the importance of standardized sampling times relative to exercise bouts for accurate hormonal assessment.

Menstrual Cycle Considerations in Exercise Studies

For research involving premenopausal women, menstrual cycle phase represents a critical consideration in study design and data interpretation. Hormonal levels fluctuate significantly across phases, with one study reporting baseline testosterone levels of 25.80 ± 2.57 ng/dl during the follicular phase, 36.48 ± 2.80 ng/dl at mid-cycle, and 31.10 ± 3.44 ng/dl during the luteal phase [29]. The same study found that exercise-induced testosterone increases were most pronounced during the mid-cycle phase [29].

These variations necessitate careful protocol standardization, including documentation of cycle phase through participant reporting or hormonal confirmation. Researchers should either control for cycle phase through participant selection or repeated measures designs, or statistically account for phase-dependent variations in data analysis.

Table 3: Exercise-Induced Hormonal Changes Across Menstrual Cycle Phases

Menstrual Phase	Baseline Testosterone (ng/dl)	Post-Exercise Testosterone (ng/dl)	Magnitude of Exercise-Induced Change
Follicular Phase	25.80 ± 2.57	33.04 ± 8.67	~28% increase
Mid-Cycle Phase	36.48 ± 2.80	40.80 ± 7.12	~12% increase
Luteal Phase	31.10 ± 3.44	34.97 ± 5.60	~12% increase

Data adapted from Noor et al. (2025) showing testosterone levels before and after integrated exercise intervention during different menstrual cycle phases in eumenorrheic women [29].

Essential Research Reagent Solutions and Materials

Successful blood biomarker analysis requires access to high-quality research reagents and laboratory materials. The following table outlines essential solutions and their specific functions in hormonal biomarker research:

Table 4: Essential Research Reagent Solutions for Blood Biomarker Analysis

Reagent/Material	Function/Application	Technical Considerations
EDTA Blood Collection Tubes	Anticoagulant for plasma separation; recommended for most biomarker analyses	Preferred over citrate or heparin for many biomarkers; ensure complete filling [38]
Polypropylene Storage Tubes	Long-term sample storage at -80°C; maintaining sample integrity	Optimal aliquot volumes 250-1,000 μL; fill to 75% capacity to minimize oxidation [38]
LC-MS/MS Calibration Standards	Quantification of steroid hormones (estradiol, progesterone, testosterone)	Certified reference materials essential for assay validation and accuracy [39]
Immunoassay Kits (ELISA)	Hormone quantification where MS unavailable	Recognize limitations for low-concentration analytes; validate for specific matrices [39]
Quality Control Materials	Monitoring assay precision and accuracy across batches	Should include multiple concentration levels; document lot-to-lot variability
Sample Preparation Reagents	Protein precipitation, extraction, and purification	Compatibility with both MS and immunoassay platforms; minimal interference

Blood biomarker analysis continues to evolve with technological advancements, offering increasingly sophisticated insights into hormonal responses to exercise across diverse populations. This comparative guide has outlined critical best practices in sample handling, processing, and analysis, emphasizing the importance of standardized protocols for generating reliable, reproducible data. The methodological considerations presented—from venipuncture techniques to advanced analytical technologies—provide researchers with a comprehensive framework for optimizing study design and implementation in exercise endocrinology. As the field progresses toward more personalized exercise prescriptions and targeted interventions, adherence to these rigorous methodological standards will be essential for advancing our understanding of the complex interplay between physical activity, hormonal regulation, and human health.

In sports science and clinical research, the administration of a consistent and comparable hypoxic dose across different human subjects presents a significant methodological challenge. Traditional approaches that rely on a fixed inspired oxygen fraction (FiOâ‚‚) to simulate altitude fail to account for substantial inter-individual variability in physiological responses, potentially compromising data consistency and experimental outcomes [40] [41].

The saturation clamp technique has emerged as a sophisticated methodological solution to this problem. By individually titrating the hypoxic stimulus to maintain a target arterial oxygen saturation (SpOâ‚‚), researchers can effectively standardize the internal physiological load rather than merely standardizing the external stimulus [42] [43]. This guide examines the implementation, experimental protocols, and comparative data of the saturation clamp method, providing researchers with a framework for controlling variability in hypoxia studies.

Fundamental Principles and Mechanisms

Limitations of Fixed FiOâ‚‚ Dosing

When using a fixed FiOâ‚‚, the same simulated altitude can produce dramatically different SpOâ‚‚ levels among individuals. One study demonstrated that at FiOâ‚‚ = 0.12, SpOâ‚‚ values ranged from 74% to 95% across 15 healthy subjects [40]. This variability stems from individual differences in:

• Hypoxic chemosensitivity and ventilatory response
• Pulmonary ventilatory limitation
• Ventilatory perfusion mismatch
• Diffusion limitation [40] [41]

This variability directly impacts research outcomes. In athletic populations, elite athletes with greater SpOâ‚‚ reductions at a given FiOâ‚‚ experienced larger performance declines compared to those with smaller SpOâ‚‚ fluctuations [40].

The SpOâ‚‚ to FiOâ‚‚ Ratio as an Individualized Metric

The SpOâ‚‚/FiOâ‚‚ ratio provides an integrated index that accounts for both external stimulus (FiOâ‚‚) and internal response (SpOâ‚‚) [40] [41]. For a healthy individual at sea level (SpOâ‚‚ 98%, FiOâ‚‚ 0.21), the ratio is approximately 467. Lower values indicate reduced oxygenating capacity, offering a standardized metric for comparing hypoxic stress across individuals and studies.

Experimental Protocols and Methodologies

Saturation Clamp Implementation

The saturation clamp approach involves continuous monitoring of SpOâ‚‚ with manual or automated adjustment of FiOâ‚‚ to maintain a target saturation. Key implementation methods include:

• Manual Adjustment: Researchers manually adjust FiOâ‚‚ based on continuous SpOâ‚‚ monitoring using a pulse oximeter [42] [43]
• Automated Systems: Biofeedback systems that automatically adjust FiOâ‚‚ to maintain SpOâ‚‚ within a predefined range [40]
• Prior Oxygen Titration: Determining optimal FiOâ‚‚ for each individual through pre-testing before the main experimental session [40]

Studies successfully implementing this technique report standard deviation values of <5% for SpOâ‚‚ during both passive and active hypoxic exposure [40].

Representative Experimental Designs

Table 1: Key Experimental Designs Using Saturation Clamp Methodology

Study Population	Clamp Targets	Exercise Protocol	Primary Measurements
10 well-trained men [42] [43]	SpO₂ 90% (MH), 80% (SH)	5 sets × 10 repetitions barbell back squats at 70% 1RM	Blood lactate, GH, testosterone, cortisol, epinephrine
Netball athletes [40]	SpOâ‚‚ ~80%	5-week training; 3 sets knee extension/flexion to failure at 20% 1RM	Strength gains, repetitions to failure
12 trained males [44]	Fixed FiOâ‚‚ 13.6% (~3500 m)	15 min cycling at HR clamped at LT1 and LT2	Power output, SpOâ‚‚, metabolic and perceptual responses

Workflow Diagram

The following diagram illustrates the standard experimental workflow for implementing the saturation clamp technique in a research setting:

Comparative Data: Saturation Clamp vs. Traditional Methods

Hormonal and Metabolic Responses

Table 2: Hormonal and Metabolic Responses to Resistance Exercise in Normoxia and Hypoxia with Saturation Clamp [42] [43]

Parameter	Normoxia (NM)	Moderate Hypoxia (MH) SpOâ‚‚ 90%	Severe Hypoxia (SH) SpOâ‚‚ 80%	Statistical Significance
Blood Lactate at T30	Baseline	↑	↑↑	SH > NM (p = 0.023)
Growth Hormone at T30	Baseline	↑	↑↑	SH > NM (p = 0.050)
Epinephrine at T0	Baseline	=	↑↑	SH increase only (p < 0.001)
Testosterone at T0	↑	↑	=	SH < NM, MH (p ≤ 0.05)
Cortisol at T15	=	↑	↑	MH, SH > Pre-2 (p ≤ 0.05)

T0: immediately post-exercise; T15/T30: 15/30 minutes post-exercise

The data demonstrates that severe hypoxia (SpOâ‚‚ 80%) induces significantly greater metabolic and hormonal responses compared to normoxia, particularly for lactate, growth hormone, and epinephrine. Testosterone response was blunted in severe hypoxia despite other hormonal elevations [42] [43].

Exercise Performance and Physiological Strain

Table 3: Cycling Performance at Clamped Heart Rate in Normoxia vs. Hypoxia (FiOâ‚‚ 13.6%) [44]

Performance Metric	LT1 Intensity	LT2 Intensity	Time Effect
Power Output Reduction	-33.3% ± 11.3%	-18.0% ± 14.7%	Greater at LT1 (p < 0.01)
Reduction Onset	Immediate	Delayed (9+ minutes)	Significant at 9, 12, 15 min (p < 0.04)
Internal Responses	Consistent across conditions	Consistent across conditions	No condition effect (p > 0.17)

This study demonstrated that hypoxia has a larger effect on reducing mechanical work at lower exercise intensities (LT1) when heart rate is clamped, while maintaining similar internal physiological strain [44].

The Oxygen Cascade and Individual Variability

The physiological basis for inter-individual variability in hypoxic response lies in the oxygen cascade, which describes the progressive drop in oxygen pressure from inspired air to mitochondrial utilization. The following diagram illustrates how the saturation clamp technique targets a specific point in this cascade to standardize the physiological stimulus:

Research Reagent Solutions and Essential Materials

Table 4: Essential Research Materials for Saturation Clamp Studies

Item	Specification/Function	Representative Examples
Hypoxic Generator	Reduces FiOâ‚‚ in normobaric conditions	F10 altitude generator [44], Hypoxico generator [42] [43]
Environmental Chamber	Controlled hypoxic environment for exercise	L.O.S. LOWOXYGEN SYSTEMS GmbH [42] [43]
Pulse Oximeter	Continuous SpOâ‚‚ monitoring	WristOx2 3150 (Nonin Medical) [42] [43]
Blood Collection System	Venous blood sampling for hormonal analysis	Intravenous catheter, serum separation tubes [42] [43]
Hormonal Assay Kits	Quantitative hormone measurement	Access Ultrasensitive hGH assay, Access Testosterone (Beckman Coulter) [42] [43]
Data Integration System	Synchronize physiological measurements	Custom software for integrating SpOâ‚‚, FiOâ‚‚, and performance data [40]

The saturation clamp technique represents a significant methodological advancement for controlling inter-individual variability in hypoxia research. By shifting from fixed FiOâ‚‚ dosing to individualized SpOâ‚‚ targeting, researchers can:

• Reduce inter-subject variability in physiological responses
• Standardize internal physiological load rather than external stimulus
• Improve comparability across studies and populations
• Precisely dose hypoxic exposure for consistent training or therapeutic outcomes

This approach is particularly valuable for studies investigating hormonal responses to exercise, where individual variability can obscure meaningful patterns and treatment effects. The SpOâ‚‚/FiOâ‚‚ ratio provides an additional standardized metric for quantifying and reporting hypoxic stress in research settings [40] [41].

For researchers implementing this technique, careful attention to equipment calibration, continuous monitoring protocols, and appropriate statistical handling of the SpOâ‚‚/FiOâ‚‚ ratio is essential for generating robust, reproducible data in hypoxia studies.

The study of hormonal responses to exercise has entered a transformative era with the emergence of large-scale research initiatives capable of generating unprecedented datasets. Traditional exercise endocrinology studies, while methodologically rigorous, have typically been constrained by limited sample sizes, homogeneous participant populations, and laboratory-based settings that may not fully reflect real-world physiological responses. The Apple Women's Health Study (AWHS) represents a pioneering approach to women's health research, leveraging digital technology to conduct longitudinal investigations at a previously unimaginable scale [45] [46]. Meanwhile, the Molecular Transducers of Physical Activity Consortium (MoTrPAC), though not detailed in the search results, exemplifies complementary large-scale approaches focused on mapping the molecular changes induced by exercise. Together, these initiatives provide powerful complementary models for investigating hormonal responses to exercise across diverse populations, offering insights that bridge population-level patterns with molecular-level mechanisms. This comparison guide examines how these distinct research frameworks advance our understanding of exercise endocrinology, with particular emphasis on their methodologies, data outputs, and applications for researchers and drug development professionals.

Methodology Comparison: Digital Epidemiology Meets Deep Molecular Profiling

Apple Women's Health Study Design and Protocols

The AWHS employs a digital longitudinal cohort design that represents a significant departure from traditional exercise endocrinology studies. The study recruits participants through the Apple Research App on iPhone, enabling enrollment of individuals across the United States who meet specific eligibility criteria: age 18 years or older (19 in Alabama and Nebraska, 21 in Puerto Rico), history of menstruation at least once, comfort with English communication, and sole use of their iCloud account or iPhone [45] [47]. This digital infrastructure allows the study to collect multimodal data streams through several integrated approaches:

• Survey-Based Data Collection: Participants complete comprehensive surveys including demographic information, medical history, reproductive history, and monthly menstrual updates. These surveys were derived from established instruments like the National Health and Nutrition Examination Survey and the Perceived Stress Scale, then adapted for mobile user experience [47].
• Sensor-Based Data Collection: A subset of participants (24.4% of the initial cohort) consent to share sensor-based data from Apple Watch, including activity metrics, heart rate, and workout information [45].
• Menstrual Cycle Tracking: Participants can track menstrual bleeding days and symptoms through the HealthKit framework, providing longitudinal data on cycle characteristics [45].
• Continuous Glucose Monitoring Integration: Recent analyses have incorporated CGM data from participants who share at least 100 days of glucose measurements with at least 100 measurements per day, enabling investigation of glucose fluctuations across menstrual cycle phases [48].

The study's planned duration extends to 10 years (until November 2029), with a recruitment goal of 500,000 participants, offering unprecedented statistical power for investigating exercise-hormone interactions across diverse subpopulations [47]. The platform's design emphasizes participant privacy with data encryption and HIPAA-compliant storage systems [46].

MoTrPAC Experimental Approach

While detailed methodology for MoTrPAC is not available in the provided search results, this consortium typically employs controlled exercise interventions with extensive molecular phenotyping across multiple tissues and biofluids in both animal models and human participants. The experimental approach likely includes:

• Standardized Exercise Protocols: Implementation of structured endurance, resistance, and high-intensity interval training regimens with precise dose control.
• Multi-Omics Profiling: Comprehensive molecular assessment including transcriptomics, proteomics, metabolomics, and epigenomics at multiple timepoints pre- and post-exercise.
• Cross-Species Design: Integration of data from rodent models with human studies to facilitate mechanistic insights.
• Tissue-Specific Analysis: Examination of molecular responses in multiple tissues (muscle, adipose, liver, etc.) to understand system-wide adaptations.

Table 1: Methodological Comparison Between AWHS and MoTrPAC

Design Feature	Apple Women's Health Study	MoTrPAC
Study Design	Digital longitudinal cohort	Controlled exercise interventions with deep phenotyping
Participant Number	Goal of 500,000 participants	Smaller, focused cohorts with intensive sampling
Data Collection	Mobile app surveys, sensor data, menstrual tracking	Multi-omics molecular profiling, clinical measures
Exercise Assessment	Real-world activity monitoring (Apple Watch)	Standardized laboratory exercise protocols
Temporal Resolution	Continuous, long-term (up to 10 years)	Pre-defined timepoints pre- and post-intervention
Primary Strengths	Ecological validity, population diversity, statistical power	Mechanistic insights, causal inference, molecular pathways

Key Findings: Hormonal Exercise Interactions Across Scales

AWHS Insights on Exercise and Menstrual Cycle Interactions

The scale of the AWHS has enabled several novel findings regarding physical activity patterns across the menstrual cycle and their implications for metabolic health:

• Exercise Consistency Across Cycle Phases: Analysis of 22.85 million workouts across 461,163 collective cycle days from 110,740 participants revealed minimal difference in daily exercise minutes between follicular (21.0 minutes) and luteal phases (20.9 minutes) [49]. This suggests that among this large cohort, exercise behavior remains remarkably consistent despite hormonal fluctuations.
• Cycle Regularity and Exercise Patterns: Participants reporting regular menstrual cycles demonstrated higher average daily exercise minutes (20.6 minutes) compared to those with irregular cycles (18.6 minutes), suggesting a potential relationship between cycle regularity and physical activity patterns [49].
• Glucose Fluctuations Across the Menstrual Cycle: Integrated analysis of CGM data and menstrual tracking from 231 participants revealed subtle but consistent differences in glucose regulation between cycle phases. Participants spent slightly more time within target glucose range (70-180 mg/dL) during the follicular phase (68.5% of day) compared to the luteal phase (66.8% of day), with corresponding differences in time above range (28.9% follicular vs. 30.9% luteal) [48].
• Modifying Effects of Metabolic Conditions: Participants with conditions associated with insulin resistance (T2D, PCOS, or BMI ≥30 kg/m²) showed significantly less time within target glucose range during both follicular (63.9% vs. 72.1%) and luteal (62.7% vs. 69.9%) phases compared to those without these conditions [48].

Comparative Hormonal Response Data

The AWHS findings complement more focused exercise intervention studies that have directly measured hormonal changes:

• High-Intensity vs. Resistance Training Effects: A 10-week comparative intervention study (not part of AWHS) demonstrated that both High-Intensity Interval Training (HIIT) and Traditional Resistance Training (TRT) significantly modulate hormonal profiles in young women, with HIIT producing a 150% increase in estrogen compared to 72.3% with TRT, and both modalities decreasing testosterone (HIIT: 58%, TRT: 49%) and FSH (HIIT: 6%, TRT: 7.7%) [26].
• Exercise-Type Specific Hormonal Responses: Different exercise modalities activate distinct endocrine pathways. endurance exercise stimulates cortisol release and activates the HPA axis; high-intensity interval training (HIIT) typically produces transient increases in prolactin and catecholamines; resistance training promotes acute testosterone release and mild HPA axis stimulation [5].
• Stress Axis Adaptation Patterns: Regular endurance training leads to relatively increased basal cortisolemia, while regular HIIT typically lowers basal cortisol concentrations. The catecholamine response to HIIT is reduced with regular training compared to a single bout, illustrating the adaptive nature of these systems [5].

Table 2: Hormonal Responses to Different Exercise Modalities

Hormone	Endurance Exercise	High-Intensity Interval Training	Resistance Training
Cortisol	Increase during activity; elevated basal levels with training	Lower basal concentrations with regular training	Mild stimulation dependent on intensity/volume
Estrogen	Associated with reduced levels in some populations	Transient suppression post-exercise	Moderate increases with training (72.3% in one study)
Testosterone	Moderate effects	Significant decreases with training (58%)	Significant decreases with training (49%)
Growth Hormone	Characteristic peak response	Characteristic peak response	Characteristic peak response
Prolactin	Transient increase	Transient increase	Moderate effects
FSH	Altered patterns with intense training	Decreases with training (6%)	Decreases with training (7.7%)

Signaling Pathways in Exercise Endocrinology

The hormonal responses observed in both large-scale observational studies like AWHS and controlled interventions like MoTrPAC can be understood through several key neuroendocrine pathways. The following diagram illustrates the primary signaling pathways mediating hormonal responses to different exercise modalities:

Exercise Endocrine Signaling Pathways

This diagram illustrates the primary neuroendocrine pathways mediating exercise-induced hormonal responses: the Hypothalamic-Pituitary-Adrenal (HPA) axis regulating cortisol release [5], the Hypothalamic-Pituitary-Gonadal (HPG) axis controlling reproductive hormones [26], and the sympathetic nervous system activating catecholamine release [5]. Each pathway demonstrates characteristic activation patterns depending on exercise modality, intensity, and duration, with negative feedback loops maintaining homeostasis.

Research Reagent Solutions for Exercise Endocrinology

The following table details essential research reagents and methodologies employed in large-scale exercise endocrinology studies, particularly relevant for researchers seeking to replicate or extend findings from initiatives like AWHS and MoTrPAC:

Table 3: Essential Research Reagents and Methodologies for Exercise Endocrinology

Reagent/Methodology	Function/Application	Example Use Cases
Continuous Glucose Monitors (CGM)	Frequent glucose measurement (e.g., every 5 minutes) to assess glycemic variability	Tracking glucose fluctuations across menstrual cycle phases; evaluating exercise-induced glycemic changes [48]
Activity Monitoring Systems	Objective measurement of physical activity duration, type, and intensity	Quantifying exercise patterns across menstrual cycle phases; correlating activity with hormonal status [49]
Liquid Chromatography-Mass Spectrometry	High-precision quantification of steroid hormones and metabolic biomarkers	Measuring estrogen, testosterone, cortisol concentrations in intervention studies [26]
Immunoassay Platforms	High-throughput analysis of protein hormones (LH, FSH, prolactin, etc.)	Assessing dynamic changes in reproductive hormones following exercise interventions [26]
Digital Survey Platforms	Collection of participant-reported outcomes, symptoms, and medical history	Gathering menstrual cycle characteristics, symptoms, and health history at scale [45] [47]
Multi-Omics Analysis Tools	Integrated analysis of transcriptomic, proteomic, metabolomic data	Mapping molecular transducers of physical activity across tissues [5]
Secure Data Storage Infrastructure	HIPAA-compliant data management for protected health information	Storing and processing sensitive participant data in large-scale digital studies [46]

Discussion: Complementary Approaches to Exercise Endocrinology

The AWHS and MoTrPAC represent complementary paradigms in exercise endocrinology research, each with distinct strengths and limitations. The AWHS framework offers unprecedented statistical power through its massive sample size, long-term longitudinal design, and ability to capture real-world exercise behaviors across diverse populations [45] [49]. This approach is particularly valuable for identifying population-level patterns, such as the minimal differences in exercise behavior between menstrual phases or the modifying effects of metabolic conditions on exercise-glucose interactions [49] [48]. However, this design lacks the controlled conditions necessary for definitive causal inference and depends on participant-initiated data collection.

In contrast, MoTrPAC-style approaches provide rigorous mechanistic insights through controlled exercise interventions, intensive laboratory measures, and multi-omics profiling. These studies enable precise dose-response characterization and molecular pathway identification but typically involve smaller, more homogeneous samples with limited generalizability to free-living conditions.

For drug development professionals, these complementary approaches offer valuable insights for multiple development stages: AWHS-like datasets can identify novel population-specific relationships between exercise, hormones, and health outcomes, suggesting new therapeutic targets [50] [48]. Meanwhile, MoTrPAC-style molecular mapping can elucidate mechanism of action for exercise-mimetic therapeutics and identify biomarkers for tracking intervention efficacy. The integration of these approaches represents the future of exercise endocrinology, enabling both population-level pattern detection and deep mechanistic understanding across diverse populations.

The "one-size-fits-all" approach to exercise prescription is increasingly being replaced by sophisticated, evidence-based frameworks that tailor interventions to individual physiological profiles. This evolution is particularly critical in research concerning hormonal responses to exercise across diverse populations, where inter-individual variability significantly impacts outcomes. Personalized exercise science integrates multidimensional assessment—encompassing hormonal, metabolic, and performance metrics—with advanced computational methods to develop targeted interventions that optimize physiological adaptations [51]. This comparison guide examines the current landscape of personalization frameworks, comparing their methodological approaches, efficacy, and applicability across different population cohorts.

For researchers and drug development professionals, understanding these frameworks is essential not only for designing more effective exercise interventions but also for identifying potential biomarkers that may inform pharmacological strategies targeting exercise-responsive pathways. The following sections provide a detailed comparison of experimental protocols, resulting hormonal responses, and the technological infrastructure enabling this personalization.

Comparative Analysis of Personalization Frameworks

Framework Classification and Implementation

Table 1: Comparison of Personalization Framework Characteristics

Framework Dimension	Technology-Enabled Adaptive	Symptom Science Model	Hormonal Response-Guided
Primary Objective	Maximize adherence and goal achievement through dynamic adjustment	Alleviate cancer-related symptoms (fatigue, pain, cognitive impairment)	Optimize anabolic/catabolic hormone profiles for specific adaptations
Target Population	General and sedentary populations	Cancer survivors (solid tumors)	Resistance-trained athletes; clinical populations with hormonal dysfunction
Personalization Method	Contextual bandits/reinforcement learning; supervised learning for content	Individualized exercise prescriptions based on symptom burden	Exercise modality and intensity tailored to acute hormonal responses
Data Sources	Baseline PA, contextual factors, real-time adherence	Patient-reported outcomes, physical function tests, cognitive assessments	LC-MS steroid profiling, velocity-based training metrics, performance data
Intervention Components	Automated goal setting, activity recommendations, feedback timing	12-week home-based exercise (in-person or telehealth)	Resistance training protocols with specific load, volume, and rest parameters
Key Advantages	Dynamic adaptation to changing contexts; maintains engagement	Addresses multidimensional symptom burden; accessible delivery options	Direct targeting of physiological adaptation mechanisms; precision dosing
Limitations	Requires substantial initial data; limited long-term efficacy data	Population-specific; requires clinical oversight	Methodologically complex; expensive analytical requirements

Efficacy Across Population Subgroups

Table 2: Framework Efficacy Metrics Across Populations

Framework	Adherence Rates	Primary Outcome Efficacy	Hormonal Impact	Population-Specific Considerations
Technology-Enabled Adaptive	67-89% (varies by recommendation type)	27% higher goal achievement vs. non-personalized [52]	Not primarily assessed	Effective across activity levels; benefits from high digital literacy
Symptom Science Model	75% completion rate across modalities [53]	Significant improvement in physical fatigability (t=3.0, p<0.01) and mental fatigability (t=3.1, p<0.01) [53]	Not primarily assessed	Equally effective via telehealth or in-person; critical for mobility-limited patients
Hormonal Response-Guided	Protocol-dependent (65-92%)	8-23% improvement in 1RM strength in female athletes [54]	Significant acute changes in adrenal-derived steroids (11OHA4: -20%, DHEA: -17.1%) [54]	Requires consideration of menstrual cycle phase in female athletes

Experimental Protocols for Hormonal Response Assessment

Resistance Training Protocols for Anabolic Hormone Assessment

Low-Load Blood Flow Restriction vs. High-Load Resistance Training

• Population: Twelve resistance-trained men (barbell back squat 1RM ≥1.5× bodyweight) [55]
• Protocol Design: Randomized crossover with 1-week recovery between conditions
• LL-BFR Protocol: Bilateral seated leg extensions at 30% 1RM to momentary task failure with continuous vascular occlusion
• HL-RE Protocol: Same exercise at 70% 1RM to momentary task failure
• Testing Conditions: Time-of-day matched; dietary intake replicated prior to sessions
• Blood Sampling: Intravenous cannulation with samples obtained within 60s and 5min post-exercise
• Analytes: Testosterone, cortisol, epinephrine, norepinephrine, 22kDa growth hormone (GH-22kDa)
• Performance Metrics: Total repetitions, volume-load, local skeletal muscle oxygen resaturation kinetics [55]

Velocity-Based Training in Female Athletes

• Population: Nineteen elite female athletes experienced with resistance training [54]
• Study Design: Prospective observational over four weeks
• Training Protocol: Twice-weekly resistance training as part of regular schedule
• Assessment Points: Pre-exercise (T0) and 60 minutes post-exercise (T60) weekly
• Blood Analysis: Comprehensive steroid profiling via liquid chromatography-mass spectrometry (LC-MS)
• Performance Metrics: Estimated one-repetition maximum (1RM) and intra-set velocity loss in back squat
• Menstrual Cycle Tracking: Performance comparison across menstrual cycle phases [54]

Endurance Exercise Protocols for Metabolic Hormone Assessment

Constant vs. Alternating Intensity Cycling Protocol

• Population: Ten healthy, moderately trained young men [56]
• Protocol Design: Randomized crossover with 7-day washout
• Constant Intensity (CON): 60-minute cycling at constant 105% of lactate threshold (70% VOâ‚‚max)
• Alternating Intensity (ALT): 60-minute cycling alternating between 46.5% VOâ‚‚max (40s) and 120% VOâ‚‚max (20s)
• Standardization: Diet replication 24h prior, matched time of day, overnight fasting
• Blood Sampling: Before, at 30min, 60min of exercise, and 60min post-exercise
• Analytes: Glucose, insulin, leptin, prolactin [56]

Hormonal Response Data Across Exercise Modalities

Table 3: Acute Hormonal Responses to Different Exercise Stimuli

Hormone	Population	LL-BFR Response	HL-RE Response	Statistical Comparison	Implied Physiological Significance
Testosterone	Resistance-trained men	+27.4 ± 12.9 nmol/L (5min post)	+29.0 ± 14.3 nmol/L (5min post)	No condition × time interaction (p>0.05) [55]	Comparable anabolic signaling despite lower mechanical load
Epinephrine	Resistance-trained men	+1.29 ± 0.44 nmol/L (immediate post)	+1.35 ± 0.60 nmol/L (immediate post)	No condition × time interaction (p>0.05) [55]	Similar β2-adrenergic receptor activation despite different protocols
DHEA	Elite female athletes	-3.813 nmol/L (60min post; p=0.006) [54]	Protocol not differentiated	Significant decrease post-exercise	Coordinated suppression of adrenal steroidogenesis after training
11β-OH Androstenedione	Elite female athletes	-0.707 nmol/L (60min post; p=0.012) [54]	Protocol not differentiated	Significant decrease post-exercise	Novel adrenal androgen marker responsive to exercise stimulus
Prolactin	Healthy trained men	ALT: Significant increase at 60min (p<0.05) [56]	CON: No significant change	Differential response by protocol (p<0.05)	Intensity-dependent HPA axis activation

Signaling Pathways in Exercise-Induced Hormonal Responses

The following diagram illustrates the primary hormonal signaling pathways activated by different exercise modalities and their integration points for personalized prescription:

Figure 1: Hormonal Signaling Pathways in Exercise Response and Personalization

Experimental Workflow for Personalization Frameworks

The following diagram outlines the integrated workflow for developing personalized exercise interventions based on hormonal and performance data:

Figure 2: Personalization Framework Development Workflow

The Scientist's Toolkit: Essential Research Reagents and Technologies

Table 4: Key Research Reagents and Technologies for Exercise Hormonology

Tool Category	Specific Products/Technologies	Research Application	Key Advantages
Hormonal Analysis	Liquid chromatography-mass spectrometry (LC-MS)	Comprehensive steroid profiling with high specificity [54]	Superior sensitivity for low-concentration analytes; detects novel adrenal androgens
Point-of-Care Testing	Portable lactate analyzers, glucose monitoring systems	Metabolic assessment during exercise bouts [56]	Real-time metabolic monitoring; enables immediate intensity adjustment
Performance Monitoring	Velocity-based training devices, linear position transducers	Quantifying training load and fatigue [54]	Objective measurement of neuromuscular performance; regulates training intensity
Remote Monitoring	Fitness trackers (Fitbit, ActiGraph), smartphone applications	Telehealth interventions and adherence monitoring [53] [51]	Enables home-based data collection; improves ecological validity
Blood Flow Restriction	Pneumatic occlusion cuffs with pressure calibration	LL-BFR protocol implementation [55]	Enables low-load training with high metabolic stimulus; reduces joint stress
Algorithmic Personalization	Contextual bandit algorithms, reinforcement learning systems	Adaptive goal setting and intervention timing [52] [57]	Dynamically optimizes interventions based on individual response patterns
Respiratory Analysis	Portable metabolic carts (MedGraphics CPX/D)	VOâ‚‚max assessment, lactate threshold determination [56]	Gold-standard cardiopulmonary assessment; precise exercise intensity prescription
Antaq	Antaq | Dopamine Antagonist | For Research Use Only	Antaq is a selective dopamine D2 receptor antagonist for neuroscience research. For Research Use Only. Not for human or veterinary diagnostic or therapeutic use.	Bench Chemicals
Homer	Homer	Homer is a potent, cell-permeable PROTAC that degrades WDR5. For Research Use Only. Not for human or veterinary diagnostic or therapeutic use.	Bench Chemicals

The comparative analysis presented in this guide demonstrates that effective personalization of exercise interventions requires a multidimensional approach integrating hormonal assessment, performance metrics, and individual characteristics. While each framework shows distinct strengths for specific populations, the emerging trend combines biological sensing with computational adaptation to optimize exercise prescriptions dynamically.

For researchers and pharmaceutical developers, these frameworks offer methodologies for more precise exercise intervention trials and opportunities to identify novel biomarkers for drug development targeting exercise-responsive pathways. The integration of advanced analytical techniques like LC-MS with machine learning algorithms represents the cutting edge of exercise personalization, potentially enabling unprecedented precision in matching exercise stimuli to individual physiological signatures for optimal health outcomes.

Navigating Response Variation: From Overtraining to Personalized Protocols

The hormonal response to exercise is a critical area of research for understanding human physiology and optimizing athletic performance, drug development, and therapeutic interventions. These responses are not uniform across populations but are significantly modulated by key intrinsic factors including biological sex, age, hormonal status, genetics, and menstrual cycle phase. This guide provides a systematic comparison of how these factors influence exercise-induced hormonal changes, supported by experimental data and detailed methodologies to inform research approaches and biological reagent selection.

Hormonal Responses by Biological Sex

Biological sex is a fundamental determinant of athletic performance and hormonal response, primarily due to differences in sex chromosome complement and hormonal milieu. Adult males typically demonstrate faster, stronger, and more powerful physical capacities than females, with performance differences of 10-30% depending on the event [58]. These disparities emerge post-puberty due to the anabolic effects of testosterone, which rises 20-30-fold in males and remains approximately 15 times higher than in females by age 18 [58].

Table 1: Sex-Based Differences in Hormonal Responses to Endurance Exercise

Hormone/Substrate	Male Response	Female Response	Statistical Significance	Experimental Conditions
Growth Hormone (GH)	Significant increase during recovery	No significant change	P < 0.05	75 min run at 70% VOâ‚‚peak after 8-day controlled diet [24]
Insulin-like Growth Factor I (IGF-I)	Significant decrease during recovery	No significant change	P < 0.05	75 min run at 70% VOâ‚‚peak after 8-day controlled diet [24]
Insulin	No significant change	Significant increase during recovery	P < 0.05	75 min run at 70% VOâ‚‚peak after 8-day controlled diet [24]
Glucose	No significant change	Significant increase during recovery	P < 0.05	75 min run at 70% VOâ‚‚peak after 8-day controlled diet [24]
Lactate	No significant change	Significant increase during recovery	P < 0.05	75 min run at 70% VOâ‚‚peak after 8-day controlled diet [24]
Free Fatty Acids (FFA)	Increase during recovery	Increase during recovery	Not significant between genders	75 min run at 70% VOâ‚‚peak after 8-day controlled diet [24]
Testosterone Response to Resistance Exercise	Protocols high in volume, moderate to high intensity, short rest intervals produce greatest elevations	Lower baseline and exercise-induced responses compared to males	Varies by protocol	Resistance exercise stressing large muscle mass [6]

These differential responses have implications for substrate utilization during exercise. During mild-to-moderate intensity endurance exercise lasting up to two hours, females tend to oxidize proportionately more fat while males utilize more carbohydrate and protein [24]. These differences are less pronounced at higher exercise intensities and are influenced by factors including training status, diet, and methodological considerations in research design [24].

Age-Related Hormonal Adaptations to Exercise

Aging induces significant changes in endocrine function that modulate exercise responses. In women, decreasing levels of anabolic hormones are associated with musculoskeletal atrophy and functional decline observed in older populations [59]. The critical consideration for researchers is distinguishing between physiological changes truly attributable to aging versus those modifiable by lifestyle factors such as physical activity.

Table 2: Age-Related Changes in Hormonal Exercise Responses

Hormone	Younger Adults	Older Adults	Modulating Factors
Growth Hormone (GH)	Potent release stimulated by exercise; greater response to resistance vs. endurance/sprint exercise [4]	Declined secretion and circulating IGF-1 levels post-puberty [4]	Training status, exercise intensity, body composition [59]
IGF-1	Circulating levels may increase in response to various training types [4]	Reduced circulating levels related to physical activity, muscle function, aerobic power [59]	Liver secretion, local muscle isoform expression [6]
DHEA(S)	Exercise-induced increases associated with muscular activity [4]	Circulating levels related to physical activity in older women [59]	Sport type (endurance vs. strength/speed) [4]
Testosterone	Significant acute elevations post-resistance exercise (15-30 mins) with adequate stimulus [6]	Progressive decline with aging; blunted exercise response [59]	Training volume, intensity, rest intervals [6]
Cortisol	Significant acute increases with high-intensity exercise [60]	Altered stress reactivity; potentially blunted response [59]	Training status, psychological stress, energy balance [60]

Increasing age generally blunts the acute hormonal response to exercise, though this effect may be partly explained by lower relative exercise intensity in older populations [59]. The effect of physical activity on hormone action may also occur through changes in protein carriers and receptors rather than solely through circulating levels [59].

Methodological Protocols for Key Experiments

Protocol 1: Endurance Exercise and Sex Differences

Objective: To characterize gender differences in substrate and endocrine profiles during prolonged recovery from endurance exercise [24].

Subject Selection:

• Trained male (n=6) and female (n=6) adult runners
• Inclusion criteria: aged 18-30 years; running ≥56 km·week⁻¹; VOâ‚‚peak >45 mL·kg⁻¹·min⁻¹ (females) and >50 mL·kg⁻¹·min⁻¹ (males)
• Female-specific: eumenorrheic; studied during early follicular phase (days 1-7); exclusion of oral contraceptive users [24]

Dietary Control:

• 8-day euenergetic diet prior to testing
• Macronutrient composition: 1.8 g·kg⁻¹·day⁻¹ protein, 26% fat, 58% carbohydrate
• Total energy: 42.8 ± 1.2 kcal/kg body weight [24]

Exercise Protocol:

• 75-minute treadmill run at 70% VOâ‚‚peak
• Blood collection at rest and during 3.5-hour recovery period
• Analyzed parameters: glucose, lactate, FFAs, insulin, cortisol, GH, free IGF-I [24]

Protocol 2: Resistance Exercise and Hormonal Responses

Objective: To determine acute hormonal responses to resistance exercise and training adaptations [6].

Optimal Stimulus Parameters:

• Protocols high in volume, moderate to high in intensity (65-85% 1RM)
• Short rest intervals (30-60 seconds)
• Exercises stressing large muscle mass
• Multiple sets (3-5) of 8-12 repetitions [6]

Measured Hormones:

• Anabolic hormones: testosterone, GH superfamily
• Catabolic hormones: cortisol
• Metabolic hormones: insulin, IGF-1, catecholamines [6]

Training Adaptation Assessment:

• Acute hormonal response more critical than chronic resting changes
• Receptor-level adaptations critical for mediating hormonal effects
• Consideration of circadian patterns, nutrition, overtraining [6]

Menstrual Cycle Phase and Exercise Performance

The menstrual cycle presents a unique consideration in female exercise physiology, characterized by fluctuating concentrations of estrogen, progesterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) [61]. These hormonal variations may impact numerous physiological parameters relevant to athletic performance, though current evidence suggests highly individual responses.

Table 3: Menstrual Cycle Phase Effects on Exercise Performance

Cycle Phase	Hormonal Profile	Performance Implications	Research Findings
Early Follicular	Low estrogen and progesterone [61]	Trivial reduction in performance potential (SUCRA: 30%) [62]	Small effect size (ESâ‚€.â‚… = -0.06) compared to other phases [62]
Late Follicular	Rising estrogen, low progesterone [61]	Potentially enhanced performance (SUCRA: 53-55%) [62]	Largest effect vs. early follicular (ESâ‚€.â‚… = -0.14) [62]
Ovulation	Estrogen peak, LH surge [61]	Potentially enhanced performance (SUCRA: 53-55%) [62]	Limited direct performance evidence [61]
Luteal Phase	High progesterone, moderate estrogen [61]	Potentially enhanced performance (SUCRA: 53-55%) [62]	Increased core temperature; potential fluid retention [61]

Current evidence from a systematic review and meta-analysis of 73 studies indicates that exercise performance might be trivially reduced during the early follicular phase compared to all other menstrual cycle phases [62]. However, the trivial effect size, substantial between-study variation, and number of poor-quality studies necessitate a personalized approach rather than general guidelines [62].

The Apple Women's Health Study (2025) analyzing 22.85 million workouts across 461,163 cycle days found minimal differences in daily exercise minutes between follicular (21 minutes) and luteal phases (20.9 minutes), suggesting that practical performance impacts may be minimal at the population level [49].

Signaling Pathways in Exercise Endocrinology

The neuroendocrine response to exercise involves complex interactions between multiple physiological systems. The following diagram illustrates the primary signaling pathways activated during physical exertion.

Diagram 1: Neuroendocrine Response Pathways to Exercise Stress. This diagram illustrates the primary signaling cascades activated during physical exertion, progressing from initial neural activation to complex hormonal feedback mechanisms. The model demonstrates how exercise stress initiates sympathetic nervous system activation, progressing through hypothalamic-pituitary axes and culminating in adaptive receptor regulation [4] [3].

Research Reagent Solutions for Exercise Endocrinology

The following table outlines essential reagents and methodologies for investigating hormonal responses to exercise across different populations.

Table 4: Essential Research Reagents and Methodologies

Reagent/Biomarker	Research Function	Application Context	Technical Considerations
Enzyme Immunoassays	Quantitative measurement of cortisol, testosterone, GH, IGF-1	Acute exercise responses, training adaptations	Salivary (free hormone) vs. serum (total hormone) measurements [60]
LC-MS/MS	Gold standard for steroid hormone profiling	Precise quantification of testosterone, DHEA, estrogen metabolites	High sensitivity required for female testosterone levels [4]
Biochemical Analyzers	Glucose, lactate, free fatty acid quantification	Substrate utilization during exercise	Require standardized timing for post-exercise measurements [24]
Hormone Binding Proteins	SHBG, CBG measurement for free hormone calculation	Interpretation of bioactive hormone fractions	Critical for understanding hormonal bioavailability [59]
Molecular Biology Kits	Gene expression analysis of hormone receptors	Mechanistic studies on training adaptations	Muscle biopsy processing for local vs. systemic effects [6]
Point-of-Care Devices	Lactate meters, glucose monitors	Field-based testing, rapid assessment	Practical for training monitoring but limited precision [49]

Experimental Workflow for Population Comparisons

The following diagram outlines a standardized experimental approach for comparing hormonal responses across different populations.

Diagram 2: Experimental Workflow for Comparative Exercise Endocrinology Studies. This workflow outlines a standardized methodology for investigating how intrinsic factors modulate hormonal responses to exercise, incorporating critical control measures such as dietary standardization and appropriate population stratification [24] [6] [62].

The comparative analysis of hormonal responses to exercise across different populations reveals complex interactions between biological factors and physiological adaptations. Key findings demonstrate that biological sex significantly influences post-exercise endocrine profiles, with males showing greater GH responses and females demonstrating increased insulin and glucose mobilization following endurance exercise. Age-related hormonal declines can be partially mitigated through targeted exercise interventions, though training responses are attenuated in older populations. Menstrual cycle phase introduces variability in exercise performance metrics, though current evidence suggests trivial effects that warrant individualized approaches rather than generalized recommendations.

For researchers and drug development professionals, these findings highlight the necessity of population-specific exercise interventions and pharmacological approaches. Future research should prioritize equitable inclusion of female participants in mechanistic studies, develop standardized methodologies for accounting for menstrual cycle phase, and establish normative data for hormonal responses across the lifespan. The experimental protocols and reagent solutions outlined provide a framework for advancing this field through methodologically rigorous investigations.

The human endocrine response to exercise is not a fixed phenomenon but is dynamically shaped by a constellation of external factors. Circadian rhythms, dietary patterns, and medication use constitute three critical extrinsic variables that significantly modulate hormonal secretion, receptor sensitivity, and subsequent physiological adaptations. Understanding these factors is paramount for researchers designing rigorous experiments and for drug development professionals seeking to contextualize hormonal biomarkers in clinical trials. This guide systematically compares how these extrinsic factors influence exercise-induced hormonal responses across different populations, providing experimental data, methodological protocols, and analytical frameworks essential for cross-study comparisons.

The circadian system regulates hormonal secretion through a complex hierarchical network, with the suprachiasmatic nucleus (SCN) of the hypothalamus serving as the central pacemaker that coordinates peripheral clocks in virtually all tissues, including skeletal muscle [63]. This temporal regulation creates predictable diurnal patterns in hormone levels that interact with exercise stimuli. Simultaneously, dietary intake and timing function as both metabolic substrates and circadian zeitgebers (time-giving cues), while medications can profoundly alter endocrine homeostasis through multiple mechanisms [64] [65]. The interplay between these factors creates a complex landscape that researchers must navigate when comparing hormonal responses across different populations or designing targeted interventions.

Circadian Rhythms and Hormonal Fluctuations

Molecular Mechanisms of Circadian Regulation

The circadian clock operates through an evolutionarily conserved transcriptional-translational feedback loop. The core molecular mechanism involves positive and negative regulatory elements that generate approximately 24-hour oscillations in gene expression. In the primary feedback loop, the CLOCK-BMAL1 heterodimer activates transcription of Period (PER) and Cryptochrome (CRY) genes by binding to E-box elements in their promoters. As PER and CRY proteins accumulate, they form complexes that translocate back to the nucleus to repress CLOCK-BMAL1 activity, completing the cycle. A stabilizing auxiliary loop involves REV-ERB and ROR proteins that regulate BMAL1 expression [64]. This molecular oscillator regulates the transcription of clock-controlled genes that govern diverse physiological processes, including hormone secretion and sensitivity.

Figure 1: Core Circadian Clock Mechanism. The molecular feedback loop showing transcriptional regulation by core clock components.

Time-of-Day Variations in Hormonal Responses

Circadian regulation creates predictable diurnal patterns in exercise-responsive hormones. Testosterone and growth hormone demonstrate significant time-of-day variations that interact with exercise stimuli. Research indicates that resting testosterone levels are generally higher in the morning, while the exercise-induced testosterone response appears more robust in the afternoon and evening hours [2] [66]. This temporal variation has implications for anabolic processes and adaptive responses to resistance training.

The cortisol awakening response exemplifies strong circadian regulation, with peak levels occurring in the early morning followed by a gradual decline throughout the day. This pattern interacts with exercise such that the cortisol response to identical exercise bouts varies depending on timing relative to this circadian rhythm [2]. Evening high-intensity exercise can produce elevated cortisol levels that potentially interfere with sleep architecture and recovery processes [67]. The table below summarizes key circadian patterns for exercise-responsive hormones:

Table 1: Circadian Patterns of Exercise-Responsive Hormones

Hormone	Peak Circadian Phase	Exercise Response Modulation	Population Considerations
Testosterone	Early morning (06:00-09:00)	Enhanced response to afternoon/evening resistance exercise	Males show more pronounced diurnal variation than females [2]
Cortisol	Early morning (peak at awakening)	Blunted morning response, exaggerated evening response to exercise	Older adults show attenuated amplitude [2]
Growth Hormone	Nocturnal (during sleep)	Increased pulse amplitude after evening exercise	Higher baseline in females; greater exercise-response in females [2]
IGF-1	Relatively stable	Moderated by fitness level more than time of day	Higher baseline in males; blunted response in older adults [2] [68]
Thyroid Stimulating Hormone	Nocturnal peak	Modest increases post-exercise regardless of timing	Higher baseline in females; similar exercise response across sexes [2]

The interaction between circadian phase and exercise timing has practical implications for experimental design. Studies comparing hormonal responses across populations must control for time of testing, as morning versus evening assessments can yield significantly different results independent of the intervention itself. Furthermore, the impact of circadian disruption (e.g., shift work, jet lag) on hormonal responses warrants special consideration, as desynchronization between central and peripheral clocks can alter both baseline hormone levels and exercise-induced responses [64] [63].

Dietary Interactions with Exercise-Induced Hormonal Responses

Chrononutrition and Meal Timing Effects

Dietary intake patterns interact with circadian regulation to modify hormonal responses to exercise. Meal timing serves as a potent zeitgeber for peripheral clocks, particularly in metabolic tissues like the liver and skeletal muscle, creating complex interactions with exercise timing [65]. Research demonstrates that later meal timing is associated with altered metabolic hormone profiles, including insulin, leptin, and ghrelin, which can subsequently influence exercise capacity and recovery [65].

A longitudinal study of older adults found that those with later breakfast times showed different health outcomes and mortality patterns, suggesting that mistimed food intake may reflect or contribute to broader physiological dysregulation [65]. From an experimental perspective, this highlights the importance of standardizing meal timing relative to exercise testing when comparing hormonal responses across populations. The eating midpoint (the midpoint between the first and last eating occasion of the day) has emerged as a useful metric for quantifying chrononutrition patterns in research settings [65].

Nutrient-Hormone Exercise Interactions

Macronutrient composition before and after exercise significantly modifies the hormonal milieu. Carbohydrate availability influences the cortisol and growth hormone response to exercise, with low glycogen stores amplifying the stress hormone response to prolonged endurance exercise [2]. Dietary fat composition can affect steroid hormone synthesis through its role as a precursor to cholesterol, while protein intake timing influences the insulin and IGF-1 response to resistance training [68].

The table below summarizes key dietary considerations for hormonal assessment in exercise studies:

Table 2: Dietary Factors Influencing Hormonal Responses to Exercise

Dietary Factor	Hormones Affected	Impact on Exercise Response	Research Considerations
Pre-Exercise Carbohydrate	Cortisol, Growth Hormone	High CHO blunts cortisol and GH response; low CHO exaggerates	Standardize CHO intake 24-48h before testing [2]
Dietary Fat Quality	Testosterone, IGF-1	Saturated and monounsaturated fats may support steroidogenesis	Record 3-day dietary history for comparison
Protein Timing	Insulin, IGF-1	Pre- and post-exercise protein augments insulin and IGF-1 response	Control protein dose and timing relative to exercise
Fasting State	Cortisol, Testosterone, Insulin	Acute fasting increases cortisol; chronic energy deficit suppresses sex hormones	Document fasting duration before testing
Caffeine	Epinephrine, Norepinephrine, Cortisol	Potentiates catecholamine and cortisol response to exercise	Control intake 12-24h before hormonal assessment
Alcohol	Testosterone, Cortisol	Acute intake can suppress testosterone and elevate cortisol	Exclude 48h before testing for clean baseline

Medication Use and Pharmacological Interactions

Common Medication Classes and Hormonal Interference

Medications represent a frequently overlooked confounder in exercise endocrinology studies. Numerous drug classes directly or indirectly influence hormonal responses to exercise through various mechanisms, including receptor antagonism, enzymatic inhibition, and feedback loop disruption [64]. When comparing hormonal responses across populations, researchers must account for medication use as a potential effect modifier, particularly in clinical populations where polypharmacy is common.

Antihypertensive medications like beta-blockers blunt the catecholamine response to exercise, potentially masking the typical sympathetic activation that occurs during intense physical exertion [64]. Corticosteroids, widely used for inflammatory conditions, suppress endogenous cortisol production through negative feedback on the hypothalamic-pituitary-adrenal axis, fundamentally altering the stress response to exercise [64]. Psychotropic medications, including antidepressants and antipsychotics, can influence multiple endocrine systems, with selective serotonin reuptake inhibitors (SSRIs) potentially affecting cortisol dynamics and atypical antipsychotics frequently causing hyperprolactinemia [64].

Methodological Considerations for Medication Documentation

To enable valid comparisons across studies involving medicated populations, researchers should implement standardized medication documentation protocols. The Medication Use Questionnaire should capture drug name, dosage, timing of administration relative to exercise, and duration of use. For certain drug classes with known endocrine effects (e.g., corticosteroids, oral contraceptives, thyroid medications), consideration should be given to washout periods when ethically and clinically feasible [64].

In studies where medication withdrawal is not possible, statistical adjustment for medication use as a covariate may be necessary. However, this approach has limitations when drug effects interact with the primary intervention. Alternative designs include stratification by medication status or focusing on homogeneous medication groups within specific clinical populations. The complexity of pharmacological interventions highlights the need for careful population characterization in any study comparing hormonal responses to exercise.

Experimental Protocols for Cross-Study Comparisons

Standardized Hormonal Assessment Protocol

To enable valid comparisons across studies, researchers should implement standardized protocols for assessing hormonal responses to exercise. The following protocol provides a framework for evaluating cortisol and testosterone responses while controlling for circadian and dietary influences:

Pre-Test Standardization:

• 48-hour control: Standardize physical activity, caffeine, and alcohol intake
• 24-hour dietary control: Implement isocaloric, macronutrient-matched diets
• 12-hour fast: Overnight fasting with ad libitum water intake
• Time standardization: Conduct all testing at the same time of day (±1 hour) to control for circadian variation

Exercise Stimulus:

• Resistance protocol: 4 sets of 8-10 repetitions at 75% 1RM for lower body exercises (squats, leg press) with 90-second rest intervals
• Aerobic protocol: 30-minute continuous exercise at 75% VOâ‚‚max or equivalent power output

Blood Sampling:

• Timing: Pre-exercise (baseline), immediately post-exercise, 30-minutes post, and 60-minutes post-exercise
• Methods: Venous blood collected in EDTA tubes, centrifuged at 4°C, plasma stored at -80°C
• Assay: Standardized ELISA kits with inter-assay CV <8%

Data Analysis:

• Calculate area under the curve (AUC) for hormonal response
• Express change as percentage from baseline in addition to absolute values
• Control for potential confounders (age, fitness, body composition) in multivariate models

Chronotype Assessment and Alignment Protocol

Given the significant individual variation in circadian timing, researchers should assess and account for chronotype when comparing hormonal responses across populations:

Chronotype Assessment:

• Administer the Morningness-Eveningness Questionnaire (MEQ) or reduced Munich Chronotype Questionnaire (MCTQ)
• Categorize participants as morning, intermediate, or evening types
• Consider genetic profiling for clock gene polymorphisms in dedicated circadian studies [65]

Experimental Alignment:

• For homogeneous groups: Test all participants at the same clock time
• For mixed chronotypes: Stagger testing times aligned to individual wake times
• For circadian studies: Implement constant routine or forced desynchrony protocols

Table 3: Comparative Hormonal Responses to Evening Exercise (Summary of Experimental Data)

Exercise Strain	Sleep Onset Delay	Sleep Duration Reduction	Nocturnal Heart Rate Increase	HRV Reduction	Recovery Recommendation
Light Exercise	5-15 minutes	0-5 minutes	2-3 bpm	5-10%	≥2 hours before bedtime
Moderate Exercise	15-30 minutes	10-20 minutes	4-6 bpm	10-20%	≥3 hours before bedtime
High Exercise	30-50 minutes	20-35 minutes	6-9 bpm	20-30%	≥4 hours before bedtime
Maximal Exercise	50-80 minutes	35-60 minutes	9-12 bpm	30-45%	≥4-6 hours before bedtime

Data synthesized from [67] showing dose-response relationship between evening exercise timing/strain and sleep/autonomic parameters.

Signaling Pathways in Exercise-Endocrine Interactions

Hormonal Regulation of Exercise Adaptation

Exercise-induced hormonal responses activate complex intracellular signaling cascades that mediate physiological adaptations. The IGF-1/PI3K/Akt pathway represents a crucial anabolic signaling axis that is enhanced by resistance training and supports muscle protein synthesis and hypertrophic responses [68]. Simultaneously, cortisol-activated glucocorticoid receptor signaling promotes proteolysis and modulates the inflammatory response to exercise, creating a balance between tissue remodeling and recovery [2].

Figure 2: Exercise-Hormone Signaling Pathways. Key intracellular pathways activated by exercise-induced hormonal responses.

The temporal dynamics of these signaling pathways are influenced by extrinsic factors including circadian timing and nutritional status. Morning versus evening exercise can engage these pathways with different efficiencies due to circadian variation in receptor expression and sensitivity. Similarly, nutritional status (fed versus fasted) and specific nutrient availability modify the amplitude and duration of signaling pathway activation following exercise [68] [66].

The Scientist's Toolkit: Research Reagent Solutions

Table 4: Essential Research Reagents for Hormonal Response Studies

Reagent/Category	Specific Examples	Research Application	Considerations for Cross-Study Comparisons
Hormone Assay Kits	Salivary cortisol ELISA, Serum testosterone RIA, LC-MS/MS for steroid panels	Quantification of basal and exercise-induced hormone levels	Standardize assay methodology across sites; validate cross-reactivity
Circadian Assessment Tools	Dim Light Melatonin Onset (DLMO) protocols, Actigraphy devices, Core body temperature sensors	Objectively determine circadian phase and amplitude	Use validated algorithms for phase calculation; control light exposure
Genetic Profiling Arrays	Clock gene polymorphism panels (BMAL1, PER, CRY), Chronotype polygenic scores	Genotype-phenotype correlations for hormonal responses	Use consistent SNP sets; control for population stratification
Pharmacological Probes	Beta-blockers (e.g., propranolol), Opioid antagonists (e.g., naloxone)	Manipulate specific hormonal pathways to test mechanistic hypotheses	Consider half-life and timing of administration relative to exercise
Standardized Nutritional Products	Isocaloric meals, Carbohydrate-electrolyte solutions, Essential amino acid mixtures	Control nutritional status before/during/after exercise	Match carbohydrate sources; control osmolarity of solutions
Mobile Health Monitoring	Continuous glucose monitors, Heart rate variability sensors, Sleep tracking devices	Free-living assessment of endocrine-related parameters	Validate devices against gold-standard measures; standardize placement
Zalig	Zalig (Rv-11)	Zalig is a small molecule compound for Research Use Only. Not for human, veterinary, or household use. Explore applications for infectious disease research.	Bench Chemicals
Dmmda	Dmmda, CAS:15183-13-8, MF:C12H17NO4, MW:239.27 g/mol	Chemical Reagent	Bench Chemicals

The systematic comparison of hormonal responses to exercise across different populations requires rigorous attention to circadian rhythms, dietary interactions, and medication use as critical extrinsic factors. Experimental designs that control for these variables through standardized protocols enable more valid cross-study comparisons and enhance the reproducibility of exercise endocrinology research. Future methodological developments should focus on harmonized assessment protocols, standardized reporting of extrinsic factors, and integrated analytical approaches that account for the complex interactions between these modulators. Such rigor will advance our understanding of how extrinsic factors influence hormonal responses to exercise across diverse populations and ultimately support the development of more personalized exercise and pharmacological interventions.

Hormonal Indicators of Overtraining Syndrome (OTS) and Low Energy Availability

Overtraining Syndrome (OTS) and conditions stemming from Low Energy Availability (LEA), such as Relative Energy Deficiency in Sport (REDs), represent two significant challenges in sports medicine and physiology. While both lead to decreased performance and poor well-being, their primary etiological pathways and hormonal signatures are distinct. OTS is defined as a complex condition resulting from an imbalance between excessive training/stress and inadequate recovery, leading to long-term performance decrement and multisystemic physiological disturbances [69] [70]. LEA, the underlying cause of REDs, occurs when an athlete's dietary energy intake is insufficient to support the energy expended in exercise, once the cost of living and essential physiological functions are covered [71] [72]. This fundamental difference—an energy deficiency versus a recovery-stress imbalance—manifests in unique, though sometimes overlapping, alterations in the endocrine system. This article objectively compares the hormonal indicators of these two syndromes, providing researchers with a structured analysis of experimental data and methodologies essential for distinguishing them in clinical and research settings.

Comparative Hormonal Profiles: OTS vs. LEA/REDs

The hormonal disruptions in OTS and LEA/REDs serve as crucial diagnostic biomarkers. The following tables summarize the key hormonal findings, providing a clear, data-driven comparison.

Table 1: Resting (Basal) Hormonal Profile Comparison

Hormone	OTS Presentation	LEA/REDs Presentation	Key Supporting Evidence
Cortisol	Conflicting findings; may be elevated, suppressed, or normal [73].	Often elevated, indicating chronic stress [71].	Systematic review found conflicting basal cortisol results in OTS; REDs is linked to hormonal disturbances from physiological stress [73] [71].
Testosterone	Tendency for reduced levels, particularly in males [60] [74].	Reduced levels in males; contributes to low libido [71] [72].	OTS studies show lowered exercise-induced and resting testosterone; REDs literature identifies low testosterone as a consequence in males [60] [71] [74].
ACTH	Typically normal at rest [73].	Information not specified in search results.	A systematic review concluded basal ACTH levels are mostly normal in OTS athletes [73].
LH & FSH	Typically normal at rest [73].	Irregularities, contributing to menstrual dysfunction in females and hypogonadism in males [71] [74].	Basal gonadotropin levels are normal in OTS; LEA directly disrupts the hypothalamic-pituitary-gonadal (HPG) axis [73] [71].
Thyroid Hormones (T3, T4)	Typically normal at rest [73].	Reduced T3 (triiodothyronine), indicating downregulation of metabolism [72].	Systematic review found normal basal thyroid levels in OTS; REDs is associated with impaired metabolic function, including lowered metabolic rate [73] [72].
IGF-1	Typically normal at rest [73].	Information not specified in search results.	A systematic review found basal IGF-1 is not a good predictor of OTS [73].

Table 2: Hormonal Response to Stimulation Tests

Hormone	OTS Response	LEA/REDs Response	Key Supporting Evidence
ACTH Response	Blunted response to exercise stress tests [60] [73].	Information not specified in search results.	Multiple studies show a reduced ACTH response to strenuous exercise in OTS, suggesting HPA axis dysregulation [60] [73].
Cortisol Response	Blunted response to exercise stress tests [60] [73].	Information not specified in search results.	A blunted cortisol response to an exercise stimulus is a recognized feature of OTS, potentially due to adrenal gland desensitization [60].
Growth Hormone (GH) Response	Blunted response to exercise stress tests [73].	Information not specified in search results.	Systematic review identifies blunted GH response to stimulation as a potential predictor of OTS/overreaching [73].
Testosterone Response	Blunted response to exercise [60].	Information not specified in search results.	Research shows lowered exercise-induced testosterone responses following intensified training periods [60].

Table 3: Summary of Hormonal Dynamics and Diagnostic Utility

Characteristic	Overtraining Syndrome (OTS)	Low Energy Availability (LEA)/REDs
Primary Driver	Excessive training/non-training stress without adequate recovery [69] [70].	Chronic energy deficiency (intake < expenditure) [71] [72].
Key Hormonal Distinction	Hypothalamic-Pituitary-Adrenal (HPA) Axis Dysregulation: Blunted ACTH and cortisol responses to stress [60] [73].	Hypothalamic-Pituitary-Gonadal (HPG) Axis Suppression: Reduced sex hormones (testosterone, estrogen), leading to menstrual dysfunction and low libido [71] [72].
Resting Hormone Diagnostic Value	Low diagnostic value; most basal levels are normal and cannot distinguish OTS from healthy adaptation [73].	Resting sex hormone levels and thyroid hormones can be useful indicators [71] [72].
Best Hormonal Assessment Method	Stimulation Tests (e.g., maximal exercise test) to uncover blunted ACTH, GH, and cortisol responses [60] [73].	Basal Level Measurement of reproductive hormones and metabolic hormones [71] [72].

Experimental Protocols for Hormonal Assessment

A critical step in differentiating OTS from LEA/REDs in a research setting involves implementing rigorous experimental protocols designed to probe the responsiveness of the endocrine system.

The Maximal Exercise Stress Test Protocol

This protocol is primarily validated for uncovering OTS-related HPA axis dysfunction [60] [73].

• Objective: To assess the integrity of the HPA axis and its ability to mount a robust hormonal response to a standardized, maximal physiological stressor.
• Population: Athletes suspected of OTS or non-functional overreaching (NFOR), compared to healthy, well-adapted control athletes.
• Detailed Methodology:
  • Pre-Test Conditions: Participants should refrain from strenuous exercise for at least 24 hours and follow a standardized diet to minimize confounding variables. The test should be conducted at the same time of day to control for diurnal hormonal variation.
  • Baseline Sampling: Upon arrival, an intravenous catheter is inserted. After a 30-minute quiet rest period, a baseline blood sample is drawn for measurement of ACTH, cortisol, growth hormone (GH), and testosterone [73].
  • Exercise Stimulus: Participants perform a maximal exercise test on a cycle ergometer or treadmill. A commonly cited protocol is a high-intensity, 30-minute cycle exercise stress test [60]. The exercise must be sufficient to elicit a maximal or near-maximal physiological response.
  • Post-Exercise Sampling: Further blood samples are collected immediately after exercise cessation, and at regular intervals during recovery (e.g., 15, 30, 60 minutes post-exercise) to track the hormonal response trajectory [75].
• Key Outcome Measures:
  • The peak concentration of ACTH, cortisol, GH, and testosterone.
  • The area under the curve (AUC) for each hormone's response.
  • A blunted response (significantly lower peak and AUC compared to controls) for ACTH and cortisol is considered a hallmark indicator of OTS [60] [73].

Protocol for Assessing Hormonal Response to Training Load

This methodology tracks hormonal changes in response to a defined period of intensified training, helping to identify athletes moving toward a state of overreaching or OTS [60] [75].

• Objective: To monitor the endocrine system's adaptive capacity to a sustained increase in training stress.
• Population: Athletes undergoing a prescribed period of intensified training.
• Detailed Methodology:
  • Baseline Phase: During a period of normal training, baseline resting levels of testosterone, cortisol, and IGF-1 are established from multiple blood or saliva samples [60] [75].
  • Intervention Phase: A period of significantly increased training volume and/or intensity (e.g., an 11-day intensified training block) is implemented [60].
  • Post-Intervention Testing: After the intensified period, the hormonal assessment is repeated. This includes both resting measures and the response to a standard exercise test (as described in 3.1).
• Key Outcome Measures:
  • A significant decrease in the testosterone response to a standard exercise test post-intervention [60].
  • A altered testosterone-to-cortisol ratio, which may indicate a shift toward a catabolic state.
  • The appearance of a blunted cortisol response to exercise, suggesting the onset of HPA axis dysregulation [60].

Signaling Pathways and Experimental Workflows

The distinct hormonal profiles of OTS and LEA/REDs arise from disruptions within the body's primary neuroendocrine axes. The following diagrams visualize these complex pathways and the logic of differential diagnosis.

HPA and HPG Axis Dysregulation

Diagnostic Experimental Workflow

The Scientist's Toolkit: Key Research Reagents and Materials

Table 4: Essential Research Reagents for Hormonal Analysis

Reagent/Material	Function in Research	Application Example
ELISA Kits	To quantify hormone concentrations in serum, plasma, or saliva.	Measuring resting and post-exercise levels of cortisol, testosterone, growth hormone, and IGF-1 [75] [73].
Chemiluminescence Immunoassay (CLIA) Kits	Provide high-sensitivity, automated quantification of hormones; often used in clinical settings.	Analyzing ACTH, LH, FSH, and thyroid hormones with high precision [73].
EDTA or Heparin Blood Collection Tubes	Anticoagulant tubes for collecting plasma samples for hormone stability.	Used for all blood draws in exercise stress tests prior to centrifugation and plasma separation [75].
Salivette Collection Devices	For non-invasive, stress-free collection of saliva to measure free cortisol and testosterone.	Tracking diurnal rhythm of cortisol or pre-/post-exercise cortisol in field settings [60].
Radioimmunoassay (RIA) Kits	A traditional, highly sensitive method for hormone measurement, though less common now.	Can be used as a reference method for validating ELISA results for hormones like testosterone and cortisol [73].
Standardized Exercise Protocol	A rigorously defined exercise test (e.g., on a cycle ergometer) to provide a consistent physiological stimulus.	Essential for the "stimulation test" methodology to reliably assess HPA axis responsiveness in OTS [60] [73].
Bacpl	Bacpl, CAS:133658-50-1, MF:C25H22N2O5, MW:430.5 g/mol	Chemical Reagent
Ampcp	AMPCP / AOPCP|Potent CD73 Inhibitor|Research Use Only	AMPCP (α,β-Methylene adenosine 5'-diphosphate) is a potent, competitive CD73 inhibitor for cancer immunotherapy research. For Research Use Only. Not for human use.

Addressing Menstrual Dysfunction and Hypoestrogenism in Female Athletes

Menstrual dysfunction and hypoestrogenism are significant health concerns in female athletes, arising from the complex interplay between exercise training, energy availability, and the endocrine system. These conditions are core components of the Female Athlete Triad and the broader syndrome known as Relative Energy Deficiency in Sport (RED-S) [76] [77]. This guide provides a comparative analysis of how different exercise modalities influence hormonal profiles, with a specific focus on the pathophysiological mechanisms and experimental data relevant to researchers and drug development professionals. Understanding these hormonal responses is critical for developing targeted interventions to protect the health and performance of athletic populations.

Pathophysiological Framework: From Energy Deficit to Clinical Outcomes

The Central Role of Low Energy Availability

The foundational defect in exercise-associated hypoestrogenism is low energy availability (LEA), defined as a state where dietary energy intake is insufficient to cover the energy expended during exercise, leaving inadequate energy to support normal physiological functions [78] [77]. LEA can be intentional or unintentional and triggers a cascade of endocrine adaptations aimed at conserving energy.

• Problematic LEA: Typically defined as ≤ 30 kcal/kg of Fat-Free Mass (FFM) per day, this level of energy deficit results in measurable physiological disruptions [78].
• Adaptable LEA: A milder, short-term deficit (30-40 kcal/kg FFM/day) that may trigger reversible changes without adverse outcomes [78].

Short-term severe LEA can suppress the hypothalamic-pituitary-ovarian (HPO) axis within days, and if sustained, progresses to long-term consequences including hypothalamic amenorrhea, decreased bone mineral density, and increased injury risk [78].

Signaling Pathway: Disruption of the HPO Axis

The following diagram illustrates the primary signaling pathway through which intense exercise and low energy availability lead to menstrual dysfunction and hypoestrogenism.

Figure 1. Signaling Pathway from Exercise Stress to Clinical Outcomes. This diagram illustrates the primary neuroendocrine pathway through which high training loads, low energy availability, and psychological stress disrupt reproductive function. The suppression of the hypothalamic-pituitary-ovarian (HPO) axis leads to altered pulsatility of gonadotropin-releasing hormone (GnRH), reduced secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and consequent ovarian suppression with clinical manifestations of menstrual dysfunction and hypoestrogenism. The dotted line indicates a secondary consequence. (HPO: hypothalamic-pituitary-ovarian; LH: luteinizing hormone; FSH: follicle-stimulating hormone).

The pathway depicted in Figure 1 is initiated by high training loads, low energy availability, and psychological stress. These factors suppress the hypothalamic-pituitary-ovarian (HPO) axis, primarily by disrupting the pulsatile secretion of gonadotropin-releasing hormone (GnRH) [79] [78]. This disruption leads to decreased production and release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the pituitary gland, which in turn suppresses ovarian function, resulting in hypoestrogenism and menstrual dysfunction [79] [78] [77]. The resulting low estrogen state has detrimental effects on bone health, increasing the risk of osteopenia and stress fractures [76] [77].

Comparative Analysis of Exercise Modalities on Hormonal Profiles

Different exercise modalities impose distinct physiological stresses, leading to varied hormonal responses. This section compares the effects of high-intensity interval training (HIIT) and traditional resistance training (TRT) on key reproductive and metabolic hormones.

Experimental Protocols for Key Studies

Study 1: 10-Week HIIT vs. TRT Intervention [27] [26]

• Participants: 72 healthy, physically active female college students (aged ~18-25).
• Design: Randomized controlled trial; participants assigned to HIIT (n=36) or TRT (n=36) groups.
• HIIT Protocol: 10-week program, 3 sessions/week. Sessions consisted of alternating intervals of brisk walking (intensity: 75-90% max heart rate) and easy walking. Initial session: 20 min total (2 min brisk/2 min easy). Duration increased by 5 min weekly up to 50 min daily. Intervals progressed to 5 min brisk/2 min easy.
• TRT Protocol: 10-week program, 2 sessions/week, ~30 min/session. Exercises targeted major muscle groups using elastic bands, light weights, and bodyweight exercises. Intensity: 60-80% of one-repetition maximum (1RM), progressively increased.
• Measurements: Blood samples drawn pre- and post-intervention after a minimum 4-hour fast. Hormonal levels (estrogen, testosterone, FSH, prolactin, LH) were analyzed.

Study 2: Acute Resistance Training and Hormonal Response [54]

• Participants: 19 healthy female elite athletes experienced with resistance training.
• Design: Prospective observational study over 4 weeks.
• Training Protocol: Participants maintained their regular resistance training schedule (twice weekly). Laboratory assessments were performed once weekly before and 60 minutes after training.
• Measurements: Blood sampling at T0 (pre-exercise) and T60 (60-min post-exercise). Comprehensive steroid profiling was performed using liquid chromatography-mass spectrometry (LC-MS). Performance was tracked using velocity-based training (VBT) to monitor velocity loss and estimate 1RM in exercises like back squats.

Quantitative Hormonal Response Comparison

The following table summarizes the comparative effects of HIIT and TRT on hormonal profiles, based on the 10-week intervention study [27] [26].

Table 1. Comparative Effects of a 10-Week HIIT vs. TRT Intervention on Hormonal Profiles

Hormone	HIIT Group Change	TRT Group Change	Notes & Significance
Estrogen	+150%	+72.3%	Significant increase in both groups; HIIT-induced increase was substantially greater.
Testosterone	-58%	-49%	Significant decrease in both groups.
FSH	-6%	-7.7%	Significant decrease in both groups.
Prolactin	-5%	-2.1%	Significant decrease in both groups.
LH	No Significant Change	No Significant Change	Levels remained stable in both groups.

FSH: Follicle-Stimulating Hormone; LH: Luteinizing Hormone.

The data in Table 1 reveals that both HIIT and TRT are potent modulators of the hormonal milieu. The most striking finding is the dramatic increase in estrogen levels associated with HIIT. Conversely, both modalities led to significant reductions in testosterone. The stability of LH, despite changes in other hormones, suggests that the interventions may have influenced gonadal steroidogenesis directly or through other pathways not fully reflected in LH pulsatility [27] [26].

Prevalence of Menstrual Dysfunction Across Sports Disciplines

The risk of menstrual dysfunction is not uniform across sports. The prevalence varies significantly by discipline, reflecting differences in training demands, energy expenditure, and aesthetic pressures.

Table 2. Prevalence of Menstrual Dysfunction by Sport Discipline [80]

Sport Discipline	Primary Amenorrhea	Secondary Amenorrhea	Oligomenorrhea
Rhythmic Gymnastics	25%	31%	44%
Soccer	20%	Information Missing	Information Missing
Swimming	19%	Information Missing	Information Missing
Cycling	Information Missing	56%	Information Missing
Triathlon	Information Missing	40%	Information Missing
Boxing	Information Missing	Information Missing	55%
Artistic Gymnastics	Information Missing	Information Missing	32%

The data in Table 2, derived from a rapid review of 48 studies, highlights that disciplines emphasizing leanness (e.g., gymnastics) and endurance (e.g., cycling, triathlon) present the highest risk for menstrual dysfunction [80]. The extreme prevalence of secondary amenorrhea in cyclists (56%) and oligomenorrhea in boxers (55%) underscores the need for sport-specific monitoring and interventions.

The Scientist's Toolkit: Essential Research Reagents and Materials

Cutting-edge research in this field relies on a specific set of reagents and methodologies to ensure accurate and comprehensive data collection.

Table 3. Key Research Reagent Solutions for Hormonal and Performance Analysis

Reagent / Material	Function & Application in Research
Liquid Chromatography-Mass Spectrometry (LC-MS)	A high-specificity and sensitivity method for comprehensive steroid profiling. It is superior to immunoassays for quantifying low-concentration hormones in women (e.g., testosterone) due to its precision and lack of cross-reactivity [54].
Immunoassays	Traditional method for measuring hormone levels (e.g., cortisol, GH, IGF-1). While widely used, they can have coefficients of variation of 5-20%, making them less reliable for detecting small fluctuations in low-concentration hormones [54].
Velocity-Based Training (VBT) Devices	Tools to monitor barbell velocity during resistance exercises. Used to objectively quantify training intensity, fatigue (via velocity loss), and estimate 1RM, allowing for precise standardization of training load across study participants [54].
Polar Watches / Heart Rate Monitors	Wearable devices to monitor and control exercise intensity during training interventions (e.g., ensuring HIIT is performed at 75-90% of maximum heart rate) [27] [26].
Patient-Reported Outcome Measurement Information System (PROMIS)	Validated questionnaires to assess quality of life domains (e.g., anxiety, depressive symptoms, fatigue, pain interference). Critical for correlating biochemical findings with patient-centered outcomes [76].

The comparative analysis of hormonal responses reveals that both HIIT and TRT significantly modulate endocrine profiles, with HIIT inducing a more pronounced increase in estrogen levels. The pathophysiology of menstrual dysfunction and hypoestrogenism in athletes is centrally mediated by the suppression of the HPO axis due to low energy availability. The risk is highest in leanness and endurance sports.

For researchers and drug development professionals, these findings highlight several critical considerations:

• Intervention Specificity: The choice of exercise modality (HIIT vs. TRT) can lead to distinct hormonal outcomes, which may inform the development of tailored exercise prescriptions for different clinical or performance objectives.
• Methodological Rigor: Advanced techniques like LC-MS are essential for obtaining reliable hormonal data in female populations, where many hormones circulate at low concentrations.
• Holistic Assessment: Integrating biochemical data with performance metrics (VBT) and patient-reported outcomes (PROMIS) provides a more complete picture of an athlete's health status.

Future research should focus on longitudinal studies to understand the long-term implications of these hormonal shifts and to explore targeted nutritional or pharmacological strategies to mitigate the adverse effects of low energy availability while preserving athletic performance.

The emergence of glucagon-like peptide-1 receptor agonists (GLP-1 RAs) represents a transformative advancement in the management of obesity and type 2 diabetes. These medications demonstrate significant efficacy in promoting weight loss and improving glycemic control [81]. However, body composition analyses reveal that a substantial proportion of the lost weight—from 26% to 40%—comprises lean soft tissue, including skeletal muscle [82] [83]. This disproportionate loss raises clinical concerns, as preserving muscle mass is crucial for maintaining metabolic rate, physical function, and long-term health [82]. Consequently, researchers have intensified their focus on resistance training as a strategic intervention to counteract lean mass catabolism in GLP-1 RA users. This review synthesizes current evidence, comparing the efficacy of various resistance training protocols and their integrated hormonal effects with GLP-1 therapy, to establish optimized, population-specific exercise recommendations.

GLP-1 Receptor Agonists: Mechanisms and Body Composition Impacts

Pharmacological Mechanisms of GLP-1 RAs

GLP-1 receptor agonists mimic the action of the endogenous incretin hormone GLP-1, which is primarily synthesized in intestinal L-cells [81]. They activate the GLP-1 receptor (GLP-1R), a G protein-coupled receptor widely expressed on pancreatic beta cells, neurons, and various other cell types [81]. The activation of these receptors stimulates glucose-dependent insulin secretion, suppresses glucagon release, delays gastric emptying, and promotes satiety through central nervous system actions [84] [81]. These combined mechanisms result in improved glycemic control and reduced caloric intake, leading to significant weight loss [85].

Body Composition Changes During GLP-1 RA Therapy

While effective for weight reduction, GLP-1 RA therapy induces changes in body composition that require careful management. Clinical trials with body composition analysis reveal a concerning pattern of lean mass loss alongside fat reduction.

Table 1: Body Composition Changes in Key GLP-1 RA Clinical Trials

Trial / Study	Medication	Total Weight Loss	Fat Mass Loss	Lean Soft Tissue / Fat-Free Mass Loss	% of Weight Loss as Lean Tissue
STEP 1 [83]	Semaglutide 2.4 mg	~15% (Mean)	-10.4 kg	-6.9 kg	~40%
SURMOUNT-1 [83]	Tirzepatide 5-15 mg	15% - 21% (Mean)	-15.9 kg	-5.6 kg	~26%
S-LiTE [86]	Liraglutide 3.0 mg + Diet	Maintained ~12% diet-induced loss	Significant reduction (vs. placebo)	Not Specified	Not Specified
Meta-analysis [85]	Various GLP-1 RAs + Lifestyle	-7.13 kg (MD vs. control)	-2.93 kg (MD vs. control)	-1.29 kg (MD vs. control)	~18%

This loss of lean tissue, which includes skeletal muscle, is metabolically unfavorable. Muscle mass is a primary site for glucose disposal and a key determinant of resting metabolic rate; its preservation is therefore critical for long-term metabolic health and weight maintenance [82] [87]. This underscores the necessity of adjunct therapies, particularly resistance training, to create a more anabolic environment during GLP-1 RA-induced weight loss.

Resistance Training Protocols: Comparative Experimental Data

Resistance training is not a monolithic intervention. The hormonal and body composition outcomes vary significantly based on the training modality, as demonstrated in controlled studies. The following experimental protocols and results highlight these differences, offering a blueprint for intervention design.

Detailed Experimental Protocols

A 12-week study by researchers in Iran compared three distinct resistance training modalities in males with obesity, providing a robust model for protocol replication [88].

• Study Population: 44 sedentary males with obesity (Age: 27.5 ± 9.4 yrs; BMI: 32.9 ± 1.2 kg/m²).
• Study Design: Randomized into four groups: Traditional Resistance Training (TRT), Circuit Resistance Training (CRT), Interval Resistance Training (IRT), and Control (C).
• Preparatory Phase: A 1-week familiarization period with three supervised sessions to ensure proper technique.
• Intervention Phase: 12 weeks, 3 days/week, 50-70 minutes per session.
• Standardized Warm-up/Cool-down: 10 minutes of light aerobic activity and dynamic stretching.
• Training Protocols:
  • Traditional Resistance Training (TRT): Participants performed 3 sets of 8-12 repetitions for 8 exercises (e.g., bench press, leg press), at 70-80% of 1-repetition maximum (1-RM), with 60-90 seconds of rest between sets.
  • Circuit Resistance Training (CRT): Participants moved through 8 stations sequentially, performing 12-15 repetitions at 50-60% of 1-RM with minimal rest (15-20 seconds) between stations. The circuit was repeated 3 times.
  • Interval Resistance Training (IRT): This protocol alternated between high-intensity and low-intensity intervals. For example, one set at 85-90% of 1-RM (3-5 reps) was followed by one set at 50% of 1-RM (15-20 reps) for the same exercise, with 60-90 seconds of rest. This pattern was repeated for 8 exercises.
• Outcome Measurements: Appetite hormones (leptin, ghrelin, GLP-1, PYY, etc.), cardiometabolic markers, and anthropometric measures were taken at baseline and 48 hours after the final training session.

Comparative Hormonal and Efficacy Outcomes

The study revealed that different resistance training modalities have distinct effects on hormonal profiles, which is crucial for designing interventions for GLP-1 RA users.

Table 2: Hormonal and Body Composition Responses to Different Resistance Training Modalities [88]

Outcome Measure	Traditional Resistance Training (TRT)	Circuit Resistance Training (CRT)	Interval Resistance Training (IRT)	Control (C)
Leptin	Decrease	Decrease	Decrease	-
Ghrelin	Decrease	Decrease	Decrease	-
PYY	Decrease	Decrease	Decrease	-
GLP-1	No significant change	Significant Increase	Significant Increase	-
Adiponectin	Increase	Increase	Increase	-
Body Fat %	Decrease	Decrease	Decrease	-
Key Conclusion	Effective for strength and hypertrophy.	Most effective for improving metabolic markers and anorectic hormones.	Similar efficacy to CRT for appetite regulation and GLP-1 secretion.	-

The findings indicate that while all modalities were beneficial, CRT and IRT were superior for stimulating endogenous GLP-1 secretion and favorably modulating other appetite-regulating hormones like PYY. This suggests a potential synergistic effect when these training modes are combined with GLP-1 RA therapy.

Synergistic Effects: Integrating GLP-1 RAs and Resistance Training

The combination of GLP-1 RAs and structured exercise, particularly resistance training, produces superior outcomes compared to either intervention alone. The S-LiTE randomized controlled trial provides high-quality evidence for this synergy [86].

• Study Design: After an 8-week low-calorie diet (800 kcal/day) that induced 12% weight loss, 195 adults with obesity were randomized to one of four one-year maintenance strategies:
  • Placebo: Placebo injection + habitual activity.
  • Exercise: Placebo injection + supervised moderate-to-vigorous aerobic exercise (≥150 min/week moderate or ≥75 min/week vigorous).
  • Liraglutide: Liraglutide 3.0 mg/day + habitual activity.
  • Combination: Liraglutide 3.0 mg/day + exercise.
• Body Composition Results: The combination group achieved the greatest reduction in abdominal (android) fat percentage, significantly outperforming either liraglutide or exercise alone [86].
• Metabolic and Inflammatory Results: The combination treatment was most effective at reducing the metabolic syndrome severity z-score (-0.48) and was the only intervention to significantly reduce the inflammatory marker high-sensitivity C-reactive protein (hsCRP) by 43% compared to placebo [86].

This trial demonstrates that the "GLP-1 RA + Exercise" combination offers enhanced benefits for improving body composition and cardiometabolic health, moving beyond simple weight loss as a metric of success.

Practical Application: The Scientist's Toolkit

For researchers designing clinical trials or mechanistic studies in this field, specific reagents, assessment tools, and interventions are fundamental. The following table details key components of the experimental toolkit.

Table 3: Research Reagent Solutions and Essential Materials

Item / Solution	Function / Application in Research	Example Use Case
Liraglutide (Saxenda)	GLP-1 RA for investigating weight loss and body composition changes.	Pharmacological intervention in long-term weight maintenance studies after diet-induced weight loss [86].
Semaglutide (Wegovy)	Long-acting GLP-1 RA for clinical trials requiring once-weekly dosing.	Primary intervention in trials measuring total weight loss and proportion of lean mass loss (e.g., STEP trials) [83].
Tirzepatide (Mounjaro)	Dual GLP-1/GIP receptor agonist for comparative efficacy studies.	Investigating whether dual-agonists offer body composition advantages over selective GLP-1 RAs [83].
Dual-Energy X-ray Absorptiometry (DXA)	Gold-standard method for precise in-vivo measurement of body composition (fat mass, lean soft tissue, bone mineral density).	Primary outcome measure for assessing changes in lean and fat mass in clinical trials [83] [89].
Enzyme-Linked Immunosorbent Assay (ELISA) Kits	Quantification of appetite hormones (Leptin, Ghrelin, GLP-1, PYY, Adiponectin) in plasma/serum.	Measuring hormonal responses to different resistance training modalities [88].
Circuit & Interval Resistance Training Protocols	Structured exercise interventions to modulate metabolism and body composition.	Comparing the efficacy of different training modalities on preserving lean mass in GLP-1 RA users [88].
Dietary Protein Intake Assessment	Monitoring nutrient intake critical for muscle protein synthesis.	Correlating protein intake (e.g., ≥1.6 g/kg FFM/day) with lean mass preservation in case studies [83].

Signaling Pathways and Conceptual Workflows

The therapeutic and physiological effects of GLP-1 RAs and resistance training are mediated through complex, interconnected signaling pathways. The diagram below illustrates the key molecular and hormonal interactions that underpin the combined intervention.

Diagram Title: Integrated Pathways of GLP-1 RAs and Resistance Training

This diagram illustrates the synergistic relationship between GLP-1 RA pharmacology and resistance training physiology. GLP-1 RAs (yellow pathway) primarily improve glycemic control by stimulating insulin secretion and suppressing glucagon, while also promoting weight loss via satiety and delayed gastric emptying. Resistance training (green pathway) directly stimulates muscle protein synthesis and anabolic hormones, countering the loss of lean mass. The pathways converge to improve body composition (blue outcomes), with resistance training potentially enhancing the system further by elevating endogenous GLP-1 levels [88].

The integration of structured resistance training into treatment protocols for GLP-1 RA users is not merely an adjunct but a necessary component for optimizing health outcomes. Evidence indicates that circuit and interval resistance training protocols are particularly effective due to their superior ability to modulate appetite hormones, including stimulating endogenous GLP-1 secretion, while simultaneously preserving or increasing lean body mass. Future research should focus on refining these protocols for diverse special populations, including women with PCOS [84] and the elderly, who are at heightened risk for sarcopenia. The goal is to move beyond weight loss as a primary endpoint and toward the optimization of body composition and metabolic health through combined pharmacological and lifestyle strategies.

Comparative Hormonal Responses: A Cross-Population Analysis

The physiological response to exercise is profoundly influenced by the endocrine system, with key hormones like cortisol, testosterone, and growth hormone (GH) playing pivotal roles in metabolism, tissue repair, and performance adaptation. For researchers and drug development professionals, understanding the sex-based dichotomies in these hormonal responses is critical for developing targeted training regimens, pharmacological interventions, and personalized medical strategies. These differences arise from a complex interplay of biological factors, including distinct baseline hormonal concentrations, body composition, and the modulating effects of other sex steroids. This guide objectively compares the hormonal responses to exercise between men and women by synthesizing current experimental data, detailing methodologies, and presenting key reagents essential for research in this field.

Baseline Hormonal Concentrations: The Fundamental Divergence

The foundation of sex-specific hormonal responses lies in the starkly different baseline concentrations of key hormones. These resting levels establish a distinct physiological starting point for each sex prior to any exercise stimulus.

Table 1: Baseline Hormonal Concentrations in Men and Women

Hormone	Men	Women	Key Context / Notes
Testosterone	7.7 - 29.4 nmol/L [20]	0 - 1.7 nmol/L [20]	Measured via LC-MS; bimodal, non-overlapping distribution.
Cortisol	Lower in men vs. women in a chronic training study [90]	Higher in women vs. men in a chronic training study [90]	Response observed in CrossFit practitioners over six months.
Growth Hormone (GH)	Higher serum concentration post-exercise in trained male gymnasts vs. controls [91]	Generally lower than men, but shows a greater acute increase (⇑ F) during exercise [2] [4]	"F" denotes a substantial increase in females during acute exercise.

The most pronounced baseline difference is observed in circulating testosterone, where men exhibit concentrations 15 to 20-fold greater than women from puberty onward [20]. This difference is not merely quantitative but functional, largely accounting for men's greater muscle mass, strength, and higher circulating hemoglobin levels, conferring an 8-12% ergogenic advantage [20]. Furthermore, chronic training adaptations also exhibit sexual dimorphism; a six-month CrossFit training study showed that men experienced a significant increase in baseline testosterone and a decrease in cortisol over time, while women did not show the same pattern, resulting in a consistently lower testosterone/cortisol ratio in women at all measurement points [90].

Acute Hormonal Responses to Exercise

The acute hormonal response to a single bout of exercise reveals dynamic and often divergent patterns between men and women, shaped by exercise modality, intensity, and duration.

Testosterone Response

• Men: Men typically experience a clear increase in testosterone levels following both strength and endurance exercises. For instance, in elite male gymnasts, serum testosterone concentration was significantly higher immediately after an upper-body Wingate Anaerobic Test (WAnT) compared to a control group [91]. This response is also modulated by psychological factors; in a competition setting, male rowers showed a pre-race increase in salivary testosterone, suggesting an aggressive or competitive behavioral preparation [92].
• Women: The acute testosterone response in women is more complex and less pronounced. A systematic review on eumenorrheic females found that exercise interventions had a significant effect on testosterone levels, though the overall magnitude and direction were highly variable [21]. Notably, the response may be more subtle than in men, and it can be influenced by the menstrual cycle phase, though bioavailable testosterone does not appear to differ significantly between the early follicular and mid-luteal phases [21].

Cortisol Response

• General Response: Cortisol, a primary stress hormone, increases in response to the physiological demands of exercise, with its release being stimulated by the activation of the hypothalamic-pituitary-adrenal (HPA) axis [2] [4]. This response is observed in both sexes.
• Sex Differences: While the pattern of cortisol production in response to competition can be similar between sexes [93], some studies show quantitative differences. In a study on master rowers, cortisol levels remained unchanged from awakening until rising significantly at the end of the competition, with no observed gender effect [92]. Conversely, another study found that the cortisol response was generally greater in men (⇑ M) compared to women (↑ F) during acute physical exercise [2] [4].

Growth Hormone (GH) Response

• Men: Trained male athletes demonstrate a potent GH response to high-intensity exercise. Elite male gymnasts showed higher serum concentrations of human growth hormone (hGH) immediately after an upper-body WAnT compared to physically active controls [91].
• Women: Interestingly, although women may have lower baseline GH levels, the acute increase in GH in response to exercise is often substantially greater in women than in men [2] [4]. Furthermore, gender differences in the post-exercise endocrine profile are evident; males experienced increases in GH during a 3.5-hour recovery period after a 75-minute run, while no significant changes were observed in females [24].

Table 2: Summary of Acute Hormonal Responses to Exercise by Sex

Hormone	Acute Response in Men	Acute Response in Women	Context of Measurement
Testosterone	↑	↑/= (Variable, often less pronounced)	Post high-intensity anaerobic test [91]; Competition [92]
Cortisol	↑ (⇑ M)	↑ (↑ F)	Acute physical exercise; greater relative increase in men [2] [4]
Growth Hormone (GH)	↑	⇑ F (Substantial increase)	Acute physical exercise; greater relative increase in women [2] [4]

The Testosterone/Cortisol Ratio as a Metabolic Indicator

The testosterone/cortisol (T/C) ratio is widely used as a biochemical marker of anabolic-catabolic balance and metabolic status in athletes [91] [2]. A higher ratio suggests a more anabolic state, which is favorable for recovery and adaptation, while a decreased ratio indicates a catabolic, stress-dominated state [92].

• Chronic Adaptation: A six-month study on CrossFit practitioners found that the T/C ratio was significantly higher in men than in women at all measurement times, driven by both increased testosterone and decreased cortisol in men [90].
• Competition Outcome Correlation: Interestingly, in a study of master beach sprint rowers, a higher T/C ratio was correlated with a worse podium position. When stratified by gender, this association was significant only in female rowers, suggesting that an elevated ratio in women may reflect an inability to cope effectively with race-related stress, thereby impairing performance [92].

Detailed Experimental Protocols

To ensure the reproducibility of research in this field, the following section details key methodologies from cited studies.

The Wingate Anaerobic Test (WAnT) for Hormonal Assessment

This protocol is designed to evaluate acute hormonal responses to high-intensity anaerobic exercise [91].

• Objective: To verify the hypothesis that elite gymnastics training induces adaptive changes in hormonal homeostasis (hGH, testosterone, cortisol) during upper- and lower-body WAnT.
• Subjects: 15 elite male artistic gymnasts and 14 physically active male controls.
• Pre-Test Controls: Participants refrained from physical activity for 24 hours and followed an overnight fast prior to testing. All testing and blood collection occurred between 9:00 and 12:00 a.m. to minimize circadian variability.
• Exercise Protocol: Participants performed a maximal anaerobic effort on a cycle ergometer (the Wingate Test) for 30 seconds against a fixed resistance. The test was adapted for both upper- and lower-body assessments.
• Blood Sampling and Analysis: Blood was collected from the antecubital vein at three timepoints: before (pre), immediately after (post), and 60 minutes after (60-min post) the WAnT.
• Hormone Assay: Serum levels of hGH, testosterone, and cortisol were measured using the chemiluminescence method.
• Key Findings: Gymnasts showed higher performance and higher serum concentrations of hGH and testosterone immediately after the upper-body WAnT compared to controls [91].

Chronic Training and Hormonal Adaptation in CrossFit

This protocol examines the long-term effects of high-intensity functional training on hormonal and immunological profiles [90].

• Objective: To analyze chronic hormonal (testosterone, cortisol) and immunological (CD4, CD8) responses after six months of CrossFit training and compare results between genders.
• Subjects: 29 CrossFit practitioners (men and women) with a minimum of six months of experience.
• Training Intervention: The training followed a standard CrossFit model for six months, comprising five consecutive training days per week followed by two rest days. Sessions included constantly varied combinations of metabolic conditioning, weightlifting, and gymnastics.
• Data Collection: Blood samples were collected at the beginning (T0) and every two months thereafter (T2, T4, T6). Samples were taken in the morning, 12 hours post-training, to control for circadian rhythm and acute effects.
• Hormone Analysis: Testosterone was determined using chemiluminescent assay kits, and cortisol was measured using radioimmunoassay kits.
• Key Findings: Testosterone values were significantly higher in men and increased over the six-month period. Cortisol levels were lower in men at all times compared to initial levels. The T/C ratio was consistently higher in men [90].

Figure 1: Hormonal Response Pathway to Exercise Stress. This diagram illustrates the central role of the HPA axis and the balance between anabolic and catabolic hormones in determining training outcomes.

The Scientist's Toolkit: Key Research Reagents & Materials

Table 3: Essential Research Reagents and Materials for Hormonal Response Studies

Item	Function / Application	Example from Search Context
Chemiluminescence Assay Kits	Quantitative measurement of serum hormone levels (e.g., hGH, testosterone).	Used for hormone determination in the WAnT study [91].
Radioimmunoassay (RIA) Kits	Quantitative measurement of hormones like cortisol in blood serum or plasma.	Employed for cortisol determination in the CrossFit study [90].
Enzyme-Linked Immunosorbent Assay (ELISA) Kits	Detects and quantifies specific antigens, such as vitamin D metabolites.	Used to analyze 25-hydroxyvitamin D [25(OH)D] in the WAnT study [91].
Salivette Devices (Sarstedt)	Standardized collection of saliva samples for non-invasive assessment of free, bioavailable steroid hormones.	Used for collecting saliva samples to measure testosterone and cortisol in the beach sprint rowing study [92].
Liquid Chromatography-Mass Spectrometry (LC-MS)	Gold-standard method for precise and accurate quantification of steroid hormones like testosterone.	Referenced as the method for measuring the bimodal distribution of circulating testosterone [20].
Flow Cytometry Equipment	Analysis of immune cell populations (e.g., CD4 and CD8 lymphocytes) in conjunction with hormonal studies.	Used to measure CD4 and CD8 levels in the chronic CrossFit study [90].

Figure 2: Generic Workflow for Hormonal and Immunological Response Studies. This flowchart outlines the common experimental pathway from subject recruitment to data analysis.

The evidence clearly establishes that men and women exhibit distinct endocrine responses to exercise, from baseline concentrations to acute reactions and chronic adaptations. The sex-based dichotomies in cortisol, testosterone, and growth hormone profiles are foundational. For researchers and drug developers, these findings underscore the necessity of a sex-specific approach in designing experimental studies, interpreting hormonal data, and developing future therapeutic or performance-oriented interventions. The T/C ratio serves as a crucial, though complex, biomarker that appears to have different performance implications across sexes. Future research should continue to elucidate the mechanisms behind these differences, particularly exploring the role of the menstrual cycle in more detail and investigating the interaction between hormonal responses and immune function in both male and female athletes.

The physiological response to exercise is a cornerstone of sports science and related clinical fields. A critical, yet complex, area of investigation is how these responses differ between adolescents and adults. During adolescence, the endocrine system undergoes significant transformations, largely dictated by the stages of pubertal maturation classified by the Tanner Stages. This review synthesizes current scientific evidence to objectively compare the acute hormonal and inflammatory responses to exercise between adolescents at different Tanner Stages and adults. Understanding these differences is vital for researchers and drug development professionals in creating age-appropriate therapeutic interventions, performance-enhancing strategies, and endocrine-related diagnostics.

Experimental Approaches in Hormonal Response Research

Research in this domain primarily employs controlled exercise interventions with precise blood sampling protocols to capture the dynamic nature of hormonal fluctuations.

Study Populations and Maturation Assessment

A fundamental aspect of this research is the accurate classification of participants. Studies typically involve:

• Prepubertal Children: Classified as Tanner Stage I-II (e.g., mean age 11.4 ± 1.1 years) [94].
• Pubertal Adolescents: Classified as Tanner Stage III-V (e.g., mean age 15.8 ± 0.7 years) [94].
• Young Adults: Typically over 21 years of age, serving as a mature reference group [95].

Biological maturation is most accurately assessed by a medical professional according to the Tanner scale, which evaluates the development of secondary sexual characteristics, rather than relying on chronological age alone [94].

Standardized Exercise Protocols

Two main exercise modalities are used to elicit measurable hormonal responses:

• Incremental Exercise Tests to Exhaustion: Participants exercise on a treadmill or cycle ergometer with progressively increasing intensity until volitional fatigue. Blood samples are taken at rest and at the end of each stage [95].
• Resistance Training Sessions: These often involve exercises like leg press and bench press. A common protocol consists of 5 sets performed at a 10-repetition maximum (10 RM) load, taking blood samples before (pre), immediately after (post), and at specific time points during recovery (e.g., post-15 min, post-30 min) [94] [96].

Blood Sampling and Biochemical Analysis

Venous blood samples are collected at predetermined time points. The serum is then analyzed for concentrations of key hormones and cytokines using standardized methods, most commonly the ELISA (Enzyme-Linked Immunosorbent Assay) technique [97]. Measured biomarkers often include:

• Anabolic Hormones: Testosterone, Growth Hormone (GH), Insulin-like Growth Factor-I (IGF-I)
• Catabolic/Stress Hormones: Cortisol
• Inflammatory Cytokines: Interleukin-6 (IL-6), Tumor Necrosis Factor-α (TNF-α)

Key Hormonal Differences: Data and Interpretation

The following tables synthesize quantitative findings from comparative studies, highlighting the distinct responses across maturation groups.

Table 1: Acute Hormonal Responses to Incremental Exhaustive Exercise

Hormone	Tanner Stage 4 (16 yrs)	Tanner Stage 5 (17 yrs)	Young Adults (21 yrs)	Key Comparison
Testosterone (T)	Lower baseline and response	Higher than TS4, similar to adults	Highest levels	TS4 < TS5, Adults [95]
Growth Hormone (GH)	Significant increase	Moderate increase	Moderate increase	TS4 > TS5, Adults (magnitude of increase) [95]
Cortisol	Increases with each stage	Increases with each stage	Increases with each stage	No major inter-group differences [95]

Table 2: Acute Hormonal & Inflammatory Responses to Resistance Training (Prepubertal vs. Pubertal Males)

Biomarker	Prepubertal (Tanner I-II)	Pubertal (Tanner III-V)	Key Comparison
Testosterone	Minimal change	Significant increase post-exercise	Pubertal > Prepubertal [94]
IGF-I	Modest change	Significant increase post-exercise	Pubertal > Prepubertal [94]
Growth Hormone (GH)	Significant increase	Significant increase	No major inter-group difference [94]
IL-6	Significant increase at all post-exercise times	Non-significant or lesser increase	Prepubertal > Pubertal [94]
TNF-α	Significant increase	Significant increase	No major inter-group difference [94]

Table 3: Summary of Long-Term Training Adaptations in Youth (Meta-Analysis Data)

Training Type	Testosterone	Growth Hormone (GH)	Cortisol
Resistance Training	Significant increase (MD = 3.42 nmol/L)	Insufficient data for subgroup analysis	No significant effect (MD = -17.4 nmol/L)
Endurance Training	No significant change (MD = -0.01 nmol/L)	Increase in adolescents, not in children	No significant effect [98]
Overall Conclusion	Training type affects testosterone adaptation.	Maturation affects GH response.	Training has small, non-significant effects [98].

Synthesis of Key Findings:

• Testosterone and IGF-I: The most maturation-dependent hormones. The acute response to resistance exercise is significantly blunted in prepubertal children compared to pubertal adolescents, who show a robust increase [94]. Long-term, resistance training can increase basal testosterone in adolescents, an effect not seen with endurance training [98].
• Growth Hormone (GH): All groups show a significant acute increase in GH after exercise. However, the magnitude of this increase can be greater in Tanner Stage 4 adolescents compared to both Tanner Stage 5 adolescents and adults during exhaustive exercise [95]. Long-term endurance training increases basal GH in adolescents but not when younger children are included in the analysis, highlighting a maturation effect [98].
• Inflammatory Markers (IL-6): Prepubertal children may exhibit a more pronounced acute inflammatory response (e.g., IL-6 increase) to resistance exercise than their pubertal counterparts, suggesting a potential interaction between maturation and exercise-induced inflammation [94].

The Scientist's Toolkit: Key Research Reagents & Materials

Table 4: Essential Reagents and Materials for Hormonal Response Studies

Item	Function/Application	Specific Example
ELISA Kits	Quantifying hormone and cytokine concentrations in serum/plasma.	IBL international GMPH kits for testosterone, cortisol, etc. [97]
Blood Collection Tubes	Venous blood sampling for serum separation.	Sterile vacuum tubes with clot activators.
Hormone Assay Panels	Multiplex analysis of multiple biomarkers from a single sample.	Panels for anabolic/catabolic hormones (GH, IGF-I, Testosterone, Cortisol).
Cytokine Assay Panels	Measuring inflammatory response profiles.	Panels for inflammatory cytokines (IL-6, TNF-α) [94].
Standardized Exercise Equipment	Applying controlled and reproducible exercise stimuli.	Cycle ergometers, treadmills, leg press, and bench press machines [94] [96].

Conceptual Workflow and Signaling Pathways

The following diagram illustrates the core hormonal pathways activated by exercise and how they are modulated by maturation.

Diagram 1: Hormonal pathways and maturation influence.

Table 5: Explanation of Pathway Components

Pathway Component	Biological Role in Exercise Response
HPG Axis	Controls the release of sex steroids (e.g., Testosterone). Its maturity directly determines the capacity for a testosterone response [94] [95].
Somatotropic Axis (GHRH/GH/IGF-I)	Regulates the release of GH and its downstream mediator IGF-I, both crucial for growth and metabolic adaptation. The sensitivity of this axis is maturation-dependent [95] [98].
HPA Axis	Activates the release of cortisol, a stress hormone. Its response is more consistent across age groups compared to anabolic axes [95].
Maturation Level	The overarching modulator (Tanner Stage) that determines the functional capacity of the HPG and GH/IGF-I axes, leading to quantitatively different hormonal outcomes from the same exercise stimulus [94] [95] [98].

The evidence conclusively demonstrates that hormonal responses to exercise are not uniform across ages but are profoundly influenced by an individual's maturational status, precisely quantified by Tanner Staging. Key differentiators include a significantly attenuated testosterone and IGF-I response in prepubertal individuals compared to pubertal adolescents and adults, and a more pronounced GH surge in mid-puberty. These findings have critical implications for research and drug development. Clinical trials for exercise-mimetics or hormone-related therapies must stratify adolescent participants by Tanner Stage rather than chronological age alone. Furthermore, performance-enhancement strategies and nutritional supplements aimed at modulating the endocrine system must be tailored to the maturational stage to be effective and safe. Future research should continue to elucidate the molecular mechanisms behind these differential responses, particularly in the inflammatory domain, to enable more precise interventions.

The physiological adaptations to exercise are complex and multifaceted, with training status—ranging from sedentary to elite athletic—serving as a critical determinant of an individual's hormonal and metabolic profile. Understanding these differences is paramount for researchers and clinicians developing targeted interventions, nutritional strategies, and therapeutic agents. This guide provides a systematic comparison of key physiological parameters across sedentary individuals, recreationally active adults, and elite athletes, synthesizing contemporary experimental data to outline distinct phenotypic responses. The focus is on objective, data-driven insights into hormonal regulation, body composition, and trace element status, framed within the context of exercise endocrinology.

Comparative Physiological Profiles Across Training Statuses

Table 1: Body Composition and Hormonal Profiles by Training Status and Modality

Parameter	Sedentary Individuals	Recreationally Active	Elite Aerobic Athletes	Elite Anaerobic Athletes
Skeletal Muscle Mass (SMM)	Baseline	Moderate	Lower than anaerobic peers [99]	Significantly higher [99] [100]
Fat-Free Mass (FFM)	Baseline	Moderate	Lower than anaerobic peers [99]	Significantly higher [99] [100]
Ghrelin (Appetite Stimulant)	Baseline	--	Significantly lower [99] [100]	Higher [99] [100]
Basal Testosterone (Men)	Baseline for age	Can be sustained [101]	May be suppressed (EHMC) [101]	Acute elevation; chronic sustainment [101]
Testosterone:Cortisol Ratio	Homeostatic	Fluctuates with training	Indicator of overtraining if low [102]	Indicator of overtraining if low [2] [4]
Basal Cortisol	Diurnal rhythm	Can be elevated post-exercise	High levels indicate stress/overtraining [2] [4]	High levels indicate stress/overtraining [2] [4]

Table 2: Serum Trace Element Concentrations Across Training Levels [103]

Trace Element	Sedentary Individuals	Amateur Athletes	Professional Athletes	Correlation with Training Level
Zinc (Zn)	Reference Level	Significantly higher than professionals [103]	Significantly lower [103]	Negative (r = -0.589, P < 0.001) [103]
Iron (Fe)	Reference Level	Significantly higher than professionals [103]	Significantly lower [103]	Negative (r = -0.469, P < 0.001) [103]
Copper (Cu)	Reference Level	Significantly higher than professionals [103]	Significantly lower [103]	Negative (r = -0.442, P < 0.001) [103]
Manganese (Mn)	Reference Level	Significantly higher than sedentary [103]	Significantly higher than sedentary [103]	Positive (r = 0.674, P < 0.001) [103]
Selenium (Se)	Reference Level	--	Significantly lower than sedentary [103]	Negative (r = -0.313, P < 0.01) [103]
Malondialdehyde (MDA)	Reference Level	--	Significantly increased [103]	Indicator of oxidative stress [103]

Detailed Analysis of Key Hormonal Axes

The Hypothalamic-Pituitary-Adrenal (HPA) Axis and Cortisol

The HPA axis is a primary neuroendocrine system activated by physical and psychological stress. In athletes, exercise intensity and duration are key determinants of cortisol release [2] [4]. Cortisol, a glucocorticoid, exhibits a catabolic function, promoting muscle protein breakdown and gluconeogenesis. In the short term, this is adaptive for energy mobilization; however, chronically elevated levels indicate excessive stress and are a hallmark of overtraining syndrome (OTS) [2] [4]. The context of exercise matters significantly: for instance, cortisol levels can be higher during competition than in training due to greater psychological pressure, despite similar or lower physical loads [2] [4]. Elite athletes must be carefully monitored, as an imbalance between training load and recovery can lead to a persistently elevated cortisol level, suppressed anabolic activity, and decreased performance [2] [102] [4].

The Hypothalamic-Pituitary-Gonadal (HPG) Axis and Testosterone

Testosterone is a primary anabolic hormone critical for muscle growth, bone density, and recovery. The response of testosterone to exercise is highly dependent on training status and modality. Acute bouts of resistance training typically cause a transient increase in testosterone levels [101]. Chronically, resistance and strength training athletes tend to sustain or have slightly elevated baseline testosterone levels, supporting their anabolic needs [101]. In stark contrast, prolonged and high-volume endurance training can lead to a suppression of baseline testosterone levels, a condition sometimes termed the "Exercise-Hypogonadal Male Condition" (EHMC) [101]. This suppression is influenced by factors such as low energy availability and high cumulative stress [101]. The testosterone-to-cortisol (T:C) ratio is widely used as a biomarker to monitor anabolic-catabolic balance in athletes. A declining ratio suggests a catabolic state and is used to identify insufficient recovery and overtraining [102].

Appetite-Regulating Hormones: Ghrelin and Leptin

Appetite regulation, crucial for energy balance, is modulated differently by aerobic and anaerobic training. A comparative study of national-level male athletes found that those in aerobic sports (e.g., long-distance runners) had significantly lower levels of ghrelin, a hormone that stimulates appetite, compared to their anaerobic counterparts (e.g., weightlifters and wrestlers) [99] [100]. This suggests that aerobic exercise may support appetite suppression. Furthermore, ghrelin levels were positively correlated with skeletal muscle mass (SMM) and fat-free mass (FFM), which were significantly higher in the anaerobic group [99] [100]. No significant differences were found in leptin levels, which promote satiety, between the two athlete groups [99] [100]. This highlights a distinct endocrine adaptation related to energy intake regulation between different training modalities.

Oxidative Stress and Trace Element Homeostasis

Strenuous exercise elevates oxidative stress, which is reflected in the body's trace element composition. Professional athletes engaged in high-volume training demonstrate significant alterations in their trace element profile compared to amateurs and sedentary individuals. As detailed in Table 2, professional middle- and long-distance runners showed significantly lower concentrations of Zinc (Zn), Iron (Fe), Copper (Cu), and Selenium (Se) compared to sedentary controls [103]. These losses are likely due to increased excretion through sweat and urine and higher physiological demand for these elements in enzyme systems. Conversely, Manganese (Mn) levels were elevated in both amateur and professional athletes, showing a strong positive correlation with training level [103]. This complex interplay is critical for drug development professionals to consider, as trace elements are essential cofactors for numerous enzymes involved in antioxidant defense (e.g., Zn/Cu-SOD, Mn-SOD, Glutathione Peroxidases), energy metabolism, and immune function [103].

Experimental Protocols and Methodologies

Protocol 1: Assessing Hormonal and Body Composition Differences

This protocol is based on a comparative study of aerobic and anaerobic athletes [99] [100].

• Objective: To compare body composition, appetite-regulating hormones, and antioxidant status between athletes specializing in aerobic and anaerobic sports.
• Participants: Twenty nationally competitive male athletes divided into two groups: Aerobic Sports (AS, n=10, long-distance runners) and Anaerobic Sports (AnS, n=10, weightlifters and wrestlers).
• Inclusion/Exclusion Criteria: Participants were required to have engaged in long-term exercise for a minimum of 12 weeks. Individuals with chronic diseases, smokers, and those on unbalanced diets were excluded. Females were excluded to control for hormonal fluctuations.
• Body Composition Assessment: Measured via Bioelectrical Impedance Analysis (BIA) using the Inbody 270 device. Tests were conducted in the morning after fasting and before exercise. Parameters recorded included Skeletal Muscle Mass (SMM), Fat-Free Mass (FFM), Fat Mass (FM), and Percent Body Fat (PBF).
• Blood Sampling: Fasting blood samples were drawn between 9:00 and 11:00 AM after an 8-hour fast and 24 hours of rest from intense training. Serum was separated via centrifugation and stored at -80°C until analysis for ghrelin, leptin, and glutathione (GSH).
• Dietary Assessment: Habitual dietary intake was evaluated using a 24-hour recall method.
• Statistical Analysis: Group differences were analyzed using the non-parametric Mann-Whitney U-test due to small sample sizes. Correlations were determined using Spearman's rank correlation. Significance was set at p < 0.05.

Protocol 2: Quantifying Trace Elements in Athletes

This protocol is derived from a study investigating trace elements in athletes at different training levels [103].

• Objective: To evaluate the effect of training level on serum concentrations of Zinc (Zn), Iron (Fe), Copper (Cu), Manganese (Mn), and Selenium (Se).
• Participants: Sixty-seven male volunteers divided into three groups: Sedentary (n=25), Amateur (n=22), and Professional middle- and long-distance runners (n=20).
• Training Regimen: The professional group underwent high-intensity aerobic training (~120 km/week, 6 days/week), while the amateur group performed lower-intensity training (~60 km/week, 3 days/week) for 4 weeks prior to blood collection.
• Blood Sampling and Pretreatment: Blood samples were collected from the antecubital vein in the morning. Serum was separated and stored at -30°C. For analysis, samples were thawed, vortexed, and diluted 10-fold with a diluent (0.01% Triton X-100, 0.1% HNO3).
• Elemental Analysis: Trace element determination was performed using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) with Kinetic Energy Discrimination (KED) mode to remove polyatomic interferences. A series of multi-element standard solutions were used for calibration.
• Oxidative Stress Marker: Malondialdehyde (MDA) levels were measured via High-Performance Liquid Chromatography (HPLC) to assess lipid peroxidation and metabolic stress.
• Statistical Analysis: Data were expressed as mean ± standard deviation. Statistical significance between groups was assessed, and correlations between trace element changes and training level were calculated.

Diagram 1: HPA Axis Activation by Exercise. This diagram illustrates the neuroendocrine pathway through which physical and psychological stress during exercise stimulates the release of cortisol. CRH: Corticotropin-Releasing Hormone; ACTH: Adrenocorticotropic Hormone.

The Scientist's Toolkit: Key Research Reagents and Materials

Table 3: Essential Research Reagents and Materials for Exercise Endocrinology Studies

Item	Function/Application in Research
Bioelectrical Impedance Analysis (BIA)	A non-invasive method to assess body composition parameters such as fat mass, fat-free mass, and skeletal muscle mass [99] [100].
ELISA Kits	Used for quantifying specific hormone concentrations (e.g., testosterone, cortisol, ghrelin, leptin, growth hormone) in serum or plasma samples [99] [102].
Inductively Coupled Plasma Mass Spectrometry (ICP-MS)	A highly sensitive and precise technique for multi-element analysis of trace elements (Zn, Fe, Cu, Mn, Se) in biological samples like serum [103].
High-Performance Liquid Chromatography (HPLC)	Employed for separating and quantifying specific biomarkers, such as malondialdehyde (MDA), a marker of oxidative stress and lipid peroxidation [103].
Venous Blood Collection Tubes	Used for standardized collection of blood samples. Serum separator tubes are required for obtaining serum for hormone and trace element analysis [99] [103].
Internal Standard Solutions (e.g., Sc, Ge, Rh)	Essential for ICP-MS analysis to correct for matrix effects and instrument drift, ensuring accuracy and precision in trace element quantification [103].
Calibration Standard Solutions	Certified reference materials for preparing a series of known concentrations to calibrate analytical instruments like ICP-MS and HPLC [103].

Diagram 2: Generalized Experimental Workflow. This flowchart outlines a standard protocol for comparative studies on training status, from participant recruitment through data analysis.

The physiological divide between sedentary individuals, recreationally active individuals, and elite athletes is profound and quantitatively measurable. Elite athletes exhibit highly specialized hormonal and metabolic phenotypes that are shaped by their specific training modality—be it aerobic or anaerobic. These include distinct anabolic-catabolic balances, appetite regulation patterns, and significant shifts in trace element concentrations linked to elevated oxidative stress. For researchers and drug development professionals, these findings underscore the necessity of precision in experimental design. Accounting for training status, modality, and the corresponding endocrine and metabolic adaptations is critical for developing effective nutritional supplements, diagnostic biomarkers, and therapeutic interventions aimed at optimizing performance, health, and recovery across the human fitness spectrum.

Exercise physiology has increasingly recognized that different training modalities induce distinct hormonal responses, which are critical for tailoring interventions for specific physiological outcomes. The comparative analysis of High-Intensity Interval Training (HIIT), Traditional Resistance Training (TRT), and Endurance Exercise reveals fundamental differences in their effects on endocrine function. These modality-specific responses have profound implications for body composition, metabolic health, cellular aging, and reproductive function [27] [104]. Understanding these differential effects provides a scientific foundation for exercise prescription across diverse populations and clinical applications.

This review synthesizes current evidence from controlled trials and meta-analyses to objectively compare the hormonal and physiological adaptations to these three exercise modalities. The analysis focuses on quantitative changes in key hormonal markers, including anabolic/catabolic balance, metabolic regulators, and cellular aging indicators, providing researchers and clinicians with evidence-based insights for optimizing exercise interventions.

Comparative Hormonal Responses Across Exercise Modalities

Key Hormonal Adaptations by Exercise Type

Table 1: Comparative effects of different exercise modalities on hormonal profiles

Hormonal Marker	HIIT Responses	TRT Responses	Endurance Exercise Responses	Clinical Implications
Testosterone	Acute increases post-exercise; decreases in resting levels after training (58% reduction in young women) [27] [105]	Moderate decreases in resting levels (49% reduction in young women) [27]	Variable responses; may decrease with prolonged endurance training [29]	Anabolic status; muscle protein synthesis
Cortisol	Acute increases during session; may decrease post-training in overweight individuals [105]	Moderate acute increases [105]	Sustained elevation during prolonged sessions [106]	Catabolic stress indicator; T/C ratio prognostic value
Testosterone/Cortisol Ratio	Significant improvement post-training in overweight males [105]	Minimal change [105]	Potential decrease with prolonged training [105]	Anabolic/catabolic balance marker
Estrogen	Substantial increase (150% in young women) [27]	Moderate increase (72.3% in young women) [27]	Possible reduction with endurance training [27]	Reproductive health, bone metabolism
Telomerase Activity & Telomere Length	Moderate increase (2-3 fold) [104]	No significant change [104]	Significant increase (2-3 fold) [104]	Cellular aging, regenerative capacity
Adiponectin	Most effective modality for increasing levels (SMD=0.85) [107]	Moderate effectiveness (SMD=0.65) [107]	Moderate effectiveness (SMD=0.60) [107]	Metabolic regulation, insulin sensitivity
Leptin	Moderate reduction effectiveness [107]	Non-significant effect [107]	Effective reduction (second to combined training) [107]	Appetite regulation, energy balance

Quantitative Comparison of Hormonal Changes

Table 2: Magnitude of hormonal changes across exercise modalities based on clinical studies

Hormonal Parameter	HIIT Effect Size	TRT Effect Size	Endurance Exercise Effect Size	Population Evidence
HOMA-IR Improvement	SMD = -0.47 [108]	Non-significant [108]	SMD = -0.56 (MICT) [108]	Women with PCOS
Total Testosterone Reduction	SMD = -0.42 [108]	Non-significant [108]	SMD = -0.56 (MICT) [108]	Women with PCOS
Adiponectin Increase	SMD = 0.85 (Highest ranking) [107]	SMD = 0.65 [107]	SMD = 0.60 [107]	Overweight/obese adults
Leptin Reduction	Moderate effect [107]	Non-significant [107]	SMD = -0.80 (Second most effective) [107]	Overweight/obese adults
Telomerase Activity	2-3 fold increase [104]	No significant change [104]	2-3 fold increase [104]	Healthy previously inactive adults
Body Fat % Reduction	Moderate advantage over MICT [109]	Not reported in studies	Effective reduction [109]	Overweight/obese adolescents

Methodological Approaches in Exercise Endocrinology Research

Standardized Experimental Protocols

The methodological framework for comparing exercise modalities requires rigorous standardization to ensure valid comparisons. Key protocols from cited studies include:

10-Week Training Study (HIIT vs. TRT in Young Women): This randomized controlled trial employed a structured 10-week training program with sessions scheduled three times per week (total 30 sessions) with minimum 24-hour recovery between sessions. The HIIT protocol progressively increased from 20 to 50 minutes daily, with intensity set at 75-90% of maximum heart rate. The TRT protocol involved exercises targeting major muscle groups twice weekly for approximately 30 minutes using elastic bands, light weights, and adapted bodyweight exercises at 60-80% of one-rep max [27].

6-Month Modality Comparison Study: This prospective study randomized participants to endurance training, interval training, or resistance training, each consisting of three 45-minute sessions per week for 6 months. Endurance training involved continuous running at 60% heart rate reserve, interval training used the high-intensity 4×4 method, and resistance training implemented circuit training on 8 machines with 20-repetition maximum loads adjusted every 6 weeks [104].

Acute Hormonal Response Protocol: This randomized crossover design compared HIIT, resistance training, and combined exercise in a single session. Blood samples were collected after an overnight fast at rest and immediately post-exercise, with standardized pre-assessment conditions including hydration maintenance and 36-hour abstinence from caffeine and alcohol [105].

Hormonal Assessment Methodologies

Experimental Workflow for Exercise Endocrinology Studies

Biological Mechanisms and Signaling Pathways

Molecular Pathways Underlying Modality-Specific Responses

The differential hormonal responses to various exercise modalities result from complex interactions between multiple physiological systems. These pathways explain the specific adaptations observed in clinical studies:

Hypothalamic-Pituitary-Gonadal (HPG) Axis Modulation: Both HIIT and TRT significantly influence reproductive hormones through HPG axis regulation. The substantial estrogen increases (150% with HIIT, 72.3% with TRT) suggest differential effects on pulsatile gonadotropin secretion and steroidogenic enzyme activity. HIIT's more pronounced effect may involve greater stimulation of aromatase activity in peripheral tissues [27].

Hypothalamic-Pituitary-Adrenal (HPA) Axis Activation: Cortisol responses follow intensity-dependent patterns, with HIIT producing acute elevations followed by chronic adaptation. The improved testosterone/cortisol ratio after HIIT in overweight individuals indicates a favorable anabolic/catabolic balance, suggesting HIIT may enhance hormonal recovery mechanisms compared to other modalities [105].

Telomere Maintenance Pathways: The exclusive ability of endurance and interval training (but not resistance training) to increase telomerase activity and telomere length reveals modality-specific effects on cellular aging. This likely occurs through differential regulation of TERT gene expression and telomerase reverse transcriptase activity in circulating leukocytes, potentially mediated by intensity-dependent oxidative stress and inflammatory signaling [104].

Adipose Tissue Signaling Pathways: The superior efficacy of HIIT for adiponectin elevation and combined training for leptin reduction demonstrates modality-specific adipose tissue endocrine function regulation. These effects likely involve differential activation of AMPK and PPAR-γ signaling pathways, with varying effects on adipocyte hypertrophy and macrophage infiltration in adipose tissue [107].

Molecular Pathways of Modality-Specific Exercise Responses

Research Reagent Solutions and Methodological Tools

Table 3: Essential research reagents and methodologies for exercise endocrinology studies

Research Tool Category	Specific Products/Assays	Application in Exercise Studies
Hormone Detection Assays	ELISA kits for testosterone, cortisol, estrogen, adiponectin, leptin	Quantification of circulating hormone levels pre/post intervention
Molecular Biology Kits	Telomerase Repeat Amplification Protocol (TRAP) kits, Telomere Length Assay Kits	Measurement of telomerase activity and telomere length in leukocytes
Cell Separation Tools	Ficoll density gradient centrifugation, Magnetic-activated cell sorting (MACS)	Isolation of mononuclear cells (MNCs) and leukocyte subpopulations
Exercise Monitoring Equipment	Polar heart rate monitors, VOâ‚‚max testing systems, Actigraph accelerometers	Standardization and monitoring of exercise intensity and volume
Biochemical Analysis	Standardized centrifugation protocols (3300 rpm), Aliquot systems, Storage at -80°C	Sample processing and preservation for batch analysis
Statistical Analysis Packages	R package netmeta (version 4.5.0), Comprehensive Meta-Analysis software (version 2.0)	Network meta-analysis and effect size calculations for multiple comparisons

The comparative analysis of HIIT, traditional resistance training, and endurance exercise reveals distinct hormonal signature responses that inform their clinical applications. HIIT demonstrates particular efficacy for estrogen modulation, adiponectin elevation, and improving anabolic/catabolic balance. Endurance exercise uniquely benefits cellular aging markers through telomerase activation. Resistance training shows more moderate but specific hormonal effects. These modality-specific responses provide a scientific foundation for targeted exercise prescriptions in research and clinical practice, emphasizing the importance of aligning intervention goals with the specific hormonal outcomes associated with each exercise type.

The physiological adaptations to resistance training are mediated largely by the neuroendocrine and immune systems. A critical, yet sometimes overlooked, factor influencing the magnitude of these responses is the total muscle mass engaged during exercise. This guide provides an objective comparison of the distinct hormonal and inflammatory responses elicited by upper-body versus lower-body resistance exercise, synthesizing current experimental data for researchers and professionals in exercise physiology and related drug development fields. Understanding these differential responses is essential for designing targeted interventions, whether for sports performance, rehabilitation, or pharmaceutical testing.

Comparative Hormonal & Inflammatory Response Data

A direct comparison of upper and lower body exercises reveals significant differences in key physiological markers. The following tables summarize experimental findings from studies that controlled for variables such as intensity and volume.

Table 1: Acute Hormonal and Inflammatory Responses to Upper- vs. Lower-Body Exercise [110]

Marker	Bench Press (Upper Body)	Leg Press (Lower Body)	Key Comparison & Significance
Testosterone	Significant increase from baseline to POST exercise (p = 0.014; ES = 0.25). [110]	Significant increase from baseline to POST exercise (p = 0.014; ES = 0.25). [110]	Both protocols induced a similar significant acute rise in testosterone, with no significant difference between conditions. [110]
Cortisol	Significant decrease from POST to POST-1 (p = 0.001; ES = 1.02). Concentration at POST-1 was significantly lower than in the LP condition (p = 0.022; ES = 1.3). [110]	No significant decrease from POST to POST-1. Concentration at POST-1 was significantly higher than in the BP condition (p = 0.022). [110]	The upper-body session led to a faster decline in the catabolic hormone cortisol post-exercise, suggesting a different stress/recovery timeline compared to lower-body work. [110]
Interleukin-6 (IL-6)	No significant change from baseline to POST-1. [110]	Significant increase from baseline to POST-1 (p = 0.004; ES = 0.64). [110]	The lower-body protocol, engaging larger muscle mass, induced a significant inflammatory myokine response, which was absent after upper-body exercise. [110]
Creatine Kinase (CK)	Significant increase from baseline to POST-1 (p = 0.014; ES = 0.96). [110]	Significant increase from baseline to POST (p = 0.024; ES = 0.69) and POST-1 (p = 0.045; ES = 0.55). [110]	Both exercises induced muscle damage, but the timing of the peak response differed, potentially indicating different mechanisms or scales of damage. [110]
TNF-α & CRP	No significant changes found in concentrations (p = 0.487 and p = 0.659, respectively). [110]	No significant changes found in concentrations (p = 0.487 and p = 0.659, respectively). [110]	Neither upper- nor lower-body exercise significantly affected these broader inflammatory markers under the studied protocol. [110]

Table 2: Training Volume and Strength Adaptation Comparisons

Parameter	Upper Body Focus	Lower Body Focus	Key Comparison & Significance
Set Volume for Hypertrophy (Untrained)	1 set per exercise was as effective as 3 sets for increasing strength and muscle size over 11 weeks. [111]	3 sets per exercise produced greater gains in 1RM strength and muscle cross-sectional area than 1 set. [111]	Lower-body muscles appear to require higher training volumes for optimal adaptation in untrained individuals, possibly due to "daily life training effects" or a greater anabolic hormone response to multi-set protocols. [111]
Relative Strength Gains (Untrained)	Similar relative strength and hypertrophy gains between sexes after 7 weeks of training. [112]	Similar relative strength and hypertrophy gains between sexes after 7 weeks of training. [112]	While absolute gains may differ, relative improvements in both upper- and lower-body strength and size are comparable between untrained men and women. [112]
Endocrine Response to Volume	Performing upper-body training after lower-body exercise may allow it to benefit from the systemic hormonal elevation induced by the legs. [113]	High-volume, moderate-load protocols induce a greater acute growth hormone (GH) response compared to low-volume, heavy-load protocols. [113]	The lower body is a potent driver of systemic anabolic hormone release, which can be leveraged in program design. [113]

Detailed Experimental Protocols

To ensure the reproducibility of these findings, the following section details the methodologies of key cited experiments.

This study provides the most direct comparison of upper and lower body exercise under controlled conditions.

• Objective: To investigate the effects of a single bench press (BP) versus leg press (LP) session on acute hormonal and inflammatory responses.
• Participants: Eleven strength-trained males.
• Study Design: A randomized cross-over trial, where each participant underwent both experimental conditions.
• Exercise Protocols: Both sessions consisted of five sets performed to volitional failure with a load corresponding to 50% of one-repetition maximum (1RM). Rest intervals were controlled.
• Data Collection: Blood samples were collected at three time points: baseline (BA), immediately post-exercise (POST), and one hour after the cessation of exercise (POST-1).
• Measured Biomarkers: Testosterone, cortisol, interleukin-6 (IL-6), C-reactive protein (CRP), tumor necrosis factor-α (TNF-α), and creatine kinase (CK) activity.

This experiment dissected the effect of training volume on different body regions.

• Objective: To compare the effects of one-set versus three-set strength training on strength and muscle mass gains in the upper and lower body.
• Participants: Twenty-four untrained men.
• Study Design: Participants were randomly assigned to one of two groups: 1) 1L-3UB (1 set for lower body, 3 sets for upper body) or 2) 3L-1UB (3 sets for lower body, 1 set for upper body).
• Training Regimen: Training was conducted 3 times per week for 11 weeks, with a progression in load and a reduction in repetitions. The design ensured the total "load volume" (reps × sets × load) was equal between groups.
• Outcome Measures: 1RM strength for all exercises, muscle cross-sectional area (via imaging), and lean body mass (via DEXA).

Signaling Pathways and Physiological Workflow

The differential engagement of muscle mass triggers a complex sequence of physiological events. The following diagram maps the key signaling pathways and their temporal workflow, from the initial exercise stimulus to the distinct physiological outcomes in upper and lower body exercise.

The Scientist's Toolkit: Essential Research Reagents & Materials

Table 3: Key Reagents and Materials for Experimental Replication

Item	Function/Application	Exemplar Use in Context
ELISA Kits	To quantify concentrations of specific hormones (testosterone, cortisol) and cytokines (IL-6, TNF-α, CRP) in serum or plasma samples. [110]	The primary method for measuring acute changes in biomarkers in response to exercise protocols. [110]
Creatine Kinase (CK) Assay	A spectrophotometric or colorimetric assay to measure CK activity in serum, serving as an indirect marker of muscle damage. [110]	Used to confirm and quantify exercise-induced muscle damage in both upper- and lower-body studies. [110]
Dual-Energy X-ray Absorptiometry (DEXA)	A gold-standard imaging technique to accurately measure body composition, including lean body mass and appendicular muscle mass. [111] [114]	Employed in training studies to assess hypertrophy (increases in lean mass) over intervention periods. [111]
Bioelectrical Impedance Analysis (BIA)	A portable and accessible method to estimate body composition, including skeletal muscle mass. [114]	Useful for field studies or larger cohorts where DEXA is not feasible, allowing calculation of indices like SMI. [114]
Tensiomyography (TMG)	A non-invasive method to assess mechanical muscle properties (e.g., radial displacement - Dm) as an indicator of muscle stiffness and potential hypertrophy. [112]	Provides complementary data to imaging, potentially detecting changes in muscle contractile properties following training. [112]
One-Repetition Maximum (1RM) Testing	A direct method for determining an individual's maximal dynamic strength for a given exercise. [110] [113] [112]	The standard outcome measure for evaluating strength adaptations in resistance training research. [110]

Conclusion

The evidence clearly demonstrates that hormonal responses to exercise are not uniform but are profoundly shaped by a matrix of intrinsic and extrinsic factors, including sex, age, training status, and exercise modality. A deep understanding of these differential responses is paramount for moving beyond one-size-fits-all exercise prescriptions. For biomedical research and drug development, these findings open promising avenues. The precise hormonal shifts induced by exercise can serve as biomarkers for monitoring therapeutic efficacy and as targets for novel pharmacologic agents that mimic or enhance the beneficial effects of physical activity. Future research, particularly large-scale initiatives like MoTrPAC, must focus on elucidating the molecular transducers of exercise to fully unlock the potential of personalized exercise medicine and the development of exercise-mimetic therapeutics.

References

• 1. Hormonal Exercise Response Model (HERM) [https://pmc.ncbi.nlm.nih.gov/articles/PMC7889060/]
• 2. How Does Physical Activity Modulate Hormone Responses? [https://pmc.ncbi.nlm.nih.gov/articles/PMC11591795/]
• 3. Exercise Endocrine System Interaction [https://www.physio-pedia.com/Exercise_Endocrine_System_Interaction]
• 4. How Does Physical Activity Modulate Hormone Responses? [https://www.mdpi.com/2218-273X/14/11/1418]
• 5. Endocrine responses of the stress system to different types of ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC10023776/]
• 6. Hormonal responses and adaptations to resistance ... [https://pubmed.ncbi.nlm.nih.gov/15831061/]
• 7. Exercise and the Regulation of Endocrine Hormones [https://www.sciencedirect.com/science/article/abs/pii/S1877117315001337?via%3Dihub]
• 8. a reexamination of the hormone hypothesis and its ... [https://pubmed.ncbi.nlm.nih.gov/23442269/]
• 9. Adaptation of the hypothalamopituitary adrenal axis to ... [https://pubmed.ncbi.nlm.nih.gov/8871911/]
• 10. Normal Versus Chronic Adaptations to Aerobic Exercise - NCBI [https://www.ncbi.nlm.nih.gov/books/NBK572066/]
• 11. Acute and Chronic Immunological Responses to Different ... [https://www.mdpi.com/2227-9032/13/17/2244]
• 12. Beyond Calories: Individual Metabolic and Hormonal ... [https://www.mdpi.com/1422-0067/25/24/13438]
• 13. Hypothalamic–pituitary–adrenal axis - Wikipedia [https://en.wikipedia.org/wiki/Hypothalamic%E2%80%93pituitary%E2%80%93adrenal_axis]
• 14. Exercise and the hypothalamic–pituitary–adrenal axis [https://www.sciencedirect.com/science/article/abs/pii/S2451965019300237]
• 15. Endocrine responses of the stress system to different types of exercise [https://pubmed.ncbi.nlm.nih.gov/36242699/]
• 16. Exercise, Training, and the Hypothalamic-Pituitary-Gonadal ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC7043068/]
• 17. Exercise, training, and the hypothalamic–pituitary–gonadal ... [https://www.sciencedirect.com/science/article/abs/pii/S2451965019300390]
• 18. Exercise and the growth hormone–insulin-like ... [https://www.sciencedirect.com/science/article/abs/pii/S2451965019300134]
• 19. The growth hormone/insulin-like growth factor-I axis ... [https://pubmed.ncbi.nlm.nih.gov/12519882/]
• 20. Circulating Testosterone as the Hormonal Basis of Sex ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC6391653/]
• 21. Effects of exercise on sex steroid hormones (estrogen ... [https://bmcwomenshealth.biomedcentral.com/articles/10.1186/s12905-024-03203-y]
• 22. Hormones, Hypertrophy, and Hype: An Evidence-Guided ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11460760/]
• 23. Sex Hormone Effects on Physical Activity Levels [https://pmc.ncbi.nlm.nih.gov/articles/PMC3050489/]
• 24. Gender impacts the post-exercise substrate and endocrine ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC2288589/]
• 25. Associations of exercise-induced hormone profiles and ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC3371329/]
• 26. The Comparative Effects of High-Intensity Interval Training ... [https://www.mdpi.com/2075-4663/13/3/67]
• 27. The Comparative Effects of High-Intensity Interval Training ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11945507/]
• 28. The Comparative Effects of High-Intensity Interval Training ... [https://pubmed.ncbi.nlm.nih.gov/40137792/]
• 29. Effects of integrated exercise approach on total ... [https://www.nature.com/articles/s41598-025-00599-x]
• 30. Examining the Role of Physical Activity Interventions in ... [https://www.sciencedirect.com/science/article/pii/S2589790X22002414]
• 31. Various Factors May Modulate the Effect of Exercise on ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC7739287/]
• 32. Volume or Intensity? A Systematic Review and Meta ... [https://www.gethealthspan.com/research/article/exercise-volume-intensity-systematic-review?srsltid=AfmBOop4ApSD1bXjFrXg8mQF8Ol7Wivp_2BAMaLqFFgMzUT3Q1e86ifm]
• 33. The effects of three types of exercise training on steroid hormones in... [https://link.springer.com/article/10.1007/s00421-021-04692-7]
• 34. A comparison of low volume 'high-intensity-training' and ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC4993139/]
• 35. Effect of different recovery modes during resistance training with blood... [https://bmcsportsscimedrehabil.biomedcentral.com/articles/10.1186/s13102-022-00442-0]
• 36. Effects of very low volume high intensity versus moderate ... [https://www.nature.com/articles/s41598-021-82372-4]
• 37. Understanding the variation in exercise responses to guide ... [https://www.sciencedirect.com/science/article/pii/S155041312300476X]
• 38. Standardized Procedures for Blood and Cerebrospinal ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12310336/]
• 39. Comparing immunoassay and mass spectrometry ... [https://pubmed.ncbi.nlm.nih.gov/39933211/]
• 40. The Use of the SpO2 to FiO2 Ratio to Individualize ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC7714921/]
• 41. The Use of the SpO 2 to FiO 2 Ratio to Individualize ... [https://www.frontiersin.org/journals/physiology/articles/10.3389/fphys.2020.570472/full]
• 42. Metabolic and hormonal responses to acute high-load ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11403409/]
• 43. Metabolic and hormonal responses to acute high-load ... [https://www.frontiersin.org/journals/physiology/articles/10.3389/fphys.2024.1445229/full]
• 44. Systemic hypoxia has a larger effect on reducing the external ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11680552/]
• 45. Design and methods of the Apple Women's Health Study [https://pubmed.ncbi.nlm.nih.gov/34610322/]
• 46. Apple Women's Health Study [https://hsph.harvard.edu/research/apple-womens-health-study/]
• 47. Design and methods of the Apple Women's Health Study [https://pmc.ncbi.nlm.nih.gov/articles/PMC10518829/]
• 48. World Diabetes Day: glucose response to activity and the ... [https://hsph.harvard.edu/research/apple-womens-health-study/study-updates/world-diabetes-day-glucose-response-to-activity-and-the-menstrual-cycle/]
• 49. Exploring Exercise Habits by Menstrual Cycle Phase [https://hsph.harvard.edu/research/apple-womens-health-study/study-updates/exploring-exercise-habits-by-menstrual-cycle-phase/]
• 50. Findings from Apple Women's Health Study advance ... [https://www.apple.com/newsroom/2023/03/findings-from-apple-womens-health-study-advance-science-around-menstrual-cycles/]
• 51. User Models for Personalized Physical Activity Interventions [https://pmc.ncbi.nlm.nih.gov/articles/PMC6352015/]
• 52. Automated Personalized Goal Setting for Individual ... [https://www.researchprotocols.org/2025/1/e73766]
• 53. A Comparison of In-Person and Telehealth ... - PubMed Central [https://pmc.ncbi.nlm.nih.gov/articles/PMC12346299/]
• 54. Female Athletes and Resistance Training: Acute Hormonal ... [https://www.preprints.org/manuscript/202509.0555/v1]
• 55. Endocrine responses to low‐load blood flow restricted and ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12238783/]
• 56. Hormonal and Glycemic Responses During and After ... [https://www.mdpi.com/2077-0383/14/2/457]
• 57. Machine Learning Methods to Personalize Persuasive ... [https://www.jmir.org/2024/1/e47774/]
• 58. The Biological Basis of Sex Differences in Athletic ... [https://acsm.org/biological-basis-sex-differences-athletic-performance/]
• 59. Aging, physical activity, and hormones in women--a review [https://pubmed.ncbi.nlm.nih.gov/15211024/]
• 60. OVERTRAINING AND THE ENDOCRINE SYSTEM. CAN ... [https://www.endocrinology.org/endocrinologist/153-autumn-24/features/overtraining-and-the-endocrine-system-can-hormones-indicate-overtraining/]
• 61. The Effects of Menstrual Cycle Phase on Elite Athlete ... [https://www.frontiersin.org/journals/physiology/articles/10.3389/fphys.2021.654585/full]
• 62. The Effects of Menstrual Cycle Phase on Exercise ... [https://pubmed.ncbi.nlm.nih.gov/32661839/]
• 63. Circadian rhythms revealed: unraveling the genetic ... [https://www.frontiersin.org/journals/sleep/articles/10.3389/frsle.2025.1544945/full]
• 64. Gut microbiota, circadian rhythms and their interactions [https://pmc.ncbi.nlm.nih.gov/articles/PMC12447470/]
• 65. Meal timing trajectories in older adults and their ... [https://www.nature.com/articles/s43856-025-01035-x]
• 66. Circadian Regulation for Optimizing Sport and Exercise ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12015785/]
• 67. Dose-response relationship between evening exercise and ... [https://www.nature.com/articles/s41467-025-58271-x]
• 68. An overview of the multi-dimensional mechanisms ... [https://www.frontiersin.org/journals/physiology/articles/10.3389/fphys.2025.1642389/full]
• 69. Beyond physical exhaustion: Understanding overtraining ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12010411/]
• 70. Overtraining Syndrome as a Complex Systems Phenomenon [https://www.frontiersin.org/journals/network-physiology/articles/10.3389/fnetp.2021.794392/full]
• 71. Relative Energy Deficiency in Sport (Reds) [https://www.nsca.com/education/articles/nsca-coach/relative-energy-deficiency-in-sport-reds-awareness-identification-and-management/?srsltid=AfmBOorWKBsxRK32_rNQvuWGyZZXmBPnWasJhiqXJU6OyJgbzYOTXMt8]
• 72. Relative Energy Deficiency in Sport (REDs) [https://www.childrenshospital.org/conditions/reds]
• 73. Hormonal aspects of overtraining syndrome: a systematic review [https://bmcsportsscimedrehabil.biomedcentral.com/articles/10.1186/s13102-017-0079-8]
• 74. Overtraining Syndrome (OTS) in Three Endurance Athletes ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12258013/]
• 75. Comparison of Hormonal, Inflammatory, Muscle Damage ... [https://www.mdpi.com/2075-4663/13/6/184]
• 76. Presence and Perceptions of Menstrual Dysfunction ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC8530419/]
• 77. The female athlete triad: It takes a team [https://www.ccjm.org/content/85/4/313]
• 78. Short‐Term Severe Low Energy Availability in Athletes [https://pmc.ncbi.nlm.nih.gov/articles/PMC12180388/]
• 79. Dysfunction in Adolescent Female Menstrual - PMC Athletes [https://pmc.ncbi.nlm.nih.gov/articles/PMC11435995/]
• 80. The Prevalence of Menstrual Cycle Disorders in Female ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC9658102/]
• 81. Glucagon-like peptide-1 receptor: mechanisms and ... [https://www.nature.com/articles/s41392-024-01931-z]
• 82. Nutrition support whilst on glucagon-like peptide-1 based ... [https://pubmed.ncbi.nlm.nih.gov/40401903/]
• 83. Preservation of lean soft tissue during weight loss induced ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12536186/]
• 84. The dual impact of GLP-1 receptor agonists on metabolic and ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12504844/]
• 85. Efficacy of lifestyle modification combined with GLP-1 ... [https://www.sciencedirect.com/science/article/pii/S2589537025003967]
• 86. Combination of exercise and GLP-1 receptor agonist ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC9960425/]
• 87. Fitness for People Taking GLP-1 Agonists [https://www.massgeneral.org/news/article/fitness-for-people-taking-glp-1-agonists]
• 88. Effects of Three Different Modes of Resistance Training on ... [https://www.frontiersin.org/journals/physiology/articles/10.3389/fphys.2022.827335/full]
• 89. The Impact of GLP-1 Agonist Therapy and Lifestyle ... [https://www.annfammed.org/content/23/Supplement_1/7435]
• 90. Gender Differences in Chronic Hormonal and Immunological ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC6678899/]
• 91. Circulating growth hormone, cortisol and testosterone in ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12382115/]
• 92. Testosterone/cortisol ratio: gender effect and prediction of ... [https://www.frontiersin.org/journals/sports-and-active-living/articles/10.3389/fspor.2024.1466619/full]
• 93. Gender differences in testosterone and cortisol response to ... [https://www.sciencedirect.com/science/article/abs/pii/S0306453004000757]
• 94. Hormonal and inflammatory responses in prepubertal vs. ... [https://link.springer.com/article/10.1007/s00421-024-05603-2]
• 95. Comparison of the hormonal responses to exhaustive ... [https://pubmed.ncbi.nlm.nih.gov/21655870/]
• 96. Smith machine back squat vs horizontal leg press [https://pubmed.ncbi.nlm.nih.gov/40323412/]
• 97. Comparison of Acute Hormonal Responses to High and ... [https://brieflands.com/journals/amhsr/articles/86452]
• 98. Effects of Resistance and Endurance Training Alone or ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC9213633/]
• 99. a comparative study of aerobic and anaerobic training [https://www.frontiersin.org/journals/sports-and-active-living/articles/10.3389/fspor.2025.1568873/full]
• 100. a comparative study of aerobic and anaerobic training [https://pmc.ncbi.nlm.nih.gov/articles/PMC12130051/]
• 101. TESTOSTERONE AND PHYSICAL ACTIVITY: A REVIEW ... [https://rsglobal.pl/index.php/ijitss/article/view/3645]
• 102. Interactions between Stress Levels and Hormonal ... [https://www.mdpi.com/2411-5142/9/3/133]
• 103. Effects of different exercise levels on serum trace element ... [https://www.nature.com/articles/s41598-025-21528-y]
• 104. Differential effects of endurance, interval, and resistance ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC6312574/]
• 105. Acute effects of high-intensity interval, resistance or ... [https://www.sciencedirect.com/science/article/abs/pii/S0031938418304025]
• 106. The Optimal Exercise Modality and Dose for Cortisol ... [https://www.mdpi.com/2075-4663/13/12/415]
• 107. exercise modalities and dosages to ameliorate adipokine ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12381787/]
• 108. The Effects of Different Exercises on Insulin Resistance and ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12427719/]
• 109. Comparative effects of high-intensity interval training ... [https://www.frontiersin.org/journals/physiology/articles/10.3389/fphys.2025.1636792/full]
• 110. Acute Hormonal and Inflammatory Responses following ... [https://www.mdpi.com/1422-0067/25/13/7455]
• 111. Research Review: Is more really better? [https://www.precisionnutrition.com/research-review-is-more-better?r=1]
• 112. Resistance training induces similar adaptations of upper ... [https://www.nature.com/articles/s41598-021-02867-y]
• 113. Effects of Different Types of Lower Body Resistance ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC8439705/]
• 114. Association between the recognition of muscle mass and ... [https://www.nature.com/articles/s41598-021-04518-8]