A review of the sports medicine literature reveals a clear underrepresentation of female athletes in research. In the current era of precision medicine, increasing attention has been directed toward the regulatory roles of estrogen and progesterone in athletic performance and health optimization. Regular fluctuations in estrogen and progesterone across menstrual cycle phases (i.e., early follicular, late follicular, and mid-luteal phases) may influence strength performance, hydration status, body composition, and energy balance. However, few studies have examined these outcomes using hormonal confirmation of menstrual cycle phases. Monophasic oral contraceptive use also represents a highly relevant hormonal condition among female athletes, as exogenous hormones suppress endogenous ovarian fluctuations and create distinct hormonal profiles across active pill-consumption and withdrawal phases. Nevertheless, the influence of oral contraceptive phases on strength-related outcomes, hydration markers, body water regulation, body composition, and energy balance remains insufficiently characterized, particularly in comparison with naturally menstruating athletes. In response to these gaps, this longitudinal observational study primarily aims to examine variations in strength-related outcomes across three distinct menstrual cycle phases (early follicular, late follicular, and mid-luteal) in eumenorrheic athletes. Secondary objectives include: (i) conducting within- and between-group comparisons of hydration status, energy balance, and strength outcomes (maximal, endurance, and explosive torque) in naturally menstruating athletes and oral contraceptive users; (ii) testing the reliability of methods used to assess body water, energy expenditure, and body composition across the menstrual cycle; (iii) exploring associations between energy availability, resting energy expenditure, and sex hormone concentrations across menstrual cycle phases; and (iv) testing, validating, and proposing methodological recommendations for the use of bioelectrical impedance analysis in tracking fluid-related changes across hormonal phases. To achieve these goals, the study will use a longitudinal observational design involving 40 female athletes, including 24 naturally menstruating athletes and 16 oral contraceptive users. Naturally menstruating athletes will be assessed during the early follicular, late follicular, and mid-luteal phases of the menstrual cycle, while oral contraceptive users will be assessed across pill-consumption and withdrawal phases. Measurements will be conducted across the three menstrual cycle phases and across oral contraceptive use phases, and will include: i) maximal voluntary isometric strength assessed using handgrip dynamometry, bench press, and leg press; ii) serum estrogen and progesterone; iii) body water and its compartments, and water turnover by dilution techniques; iv) hydration status by plasma osmolality, sodium, and vasopressin; v) energy balance by doubly labeled water and body composition changes; vi) resting energy expenditure by indirect calorimetry.

BACKGROUND:

Sports medicine has revealed a clear underrepresentation of female athletes in research, with findings from male participants often being used as a proxy for females. In the new era of precision medicine, the focus has shifted to the regulatory role of sex hormones in enhancing performance and optimizing athletic health. Sex hormone levels are relatively stable on a daily basis in men but fluctuate throughout the menstrual cycle (MC) in women. Menstrual status can range from amenorrhoea to natural menstruation (i.e., MC lasting 21-35 days), with the latter being classified as anovulatory, luteal phase-deficient, or ovulatory. The MC is divided into two main phases: the follicular phase (lasting from menstruation to ovulation) and the luteal phase (lasting from ovulation to the onset of menstruation). Regular fluctuations in sex hormones occur during an ovulatory MC, with three hormonal phases based on estrogen and progesterone fluctuations: early follicular (low levels of estrogen and progesterone), late follicular (high levels of estrogen and low levels of progesterone), and mid-luteal (high levels of estrogen and progesterone). MC duration and hormone profiles show large individual variability due to lifestyle factors or oral contraceptive (OC) use. The most commonly used are combined OCs, which lead to ovarian suppression through negative feedback from exogenous estrogen and progesterone. Estrogen may contribute to increases in muscle mass by improving intrinsic muscle quality, enabling muscle fibres to generate force, and increasing collagen levels in connective tissues, whereas progesterone is associated with protein catabolism, potentially reducing muscle strength. Adequate levels of strength are critical for improving performance, reducing injury risk, and enhancing sport-specific skills (e.g., managing body mass, running, and jumping). Maximal strength, endurance strength (fatigue-resistance-related motor tasks), and power are crucial in sports, but the variations associated with the MC are not clearly understood. These misconceptions are partly related to hormone fluctuations throughout the MC. Some authors suggest that strength performance varies across MC phases and improves when estrogen levels are higher (e.g., during the follicular phase), while others consider these variations inconclusive. Additionally, variations in fluid and energy balance due to hormonal fluctuations throughout the MC are related to muscle power and strength, but their role has yet to be explored and requires further investigation. On the other hand, low energy availability, defined as less than 30 kcal/kg fat-free mass/day in women, adversely affects the secretion of sex hormones (e.g., estrogen and progesterone) and causes functional hypothalamic amenorrhea, which decreases sarcoplasmic and myofibrillar protein synthesis in athletes, thereby reducing strength. These hormonal fluctuations and their impact on energy availability also extend to fluid balance. Fluid balance is affected because both estrogen and progesterone can influence thermoregulatory and fluid-regulatory systems, likely causing shifts in water compartments. In women, estrogen and progesterone can affect thirst, fluid intake, and sodium regulation, thereby influencing hydration status throughout the MC. Estrogen affects the threshold for vasopressin release, whereas progesterone increases aldosterone and vasopressin levels, collectively increasing fluid retention and potentially expanding extracellular water, especially during the mid-luteal phase (higher estrogen and progesterone levels). To date, no studies have investigated hydration status, including water compartment changes, across MC phases or in OC users among athletes. Hydration status affects strength and athletic health, but how fluid shifts occur throughout MC-related hormonal fluctuations remains poorly understood. Previous research has observed that intracellular water gains, as assessed by dilution techniques, improved strength and jump height over the course of an athletic season; however, neither MC phases nor OC use were considered. Although dilution techniques are considered the reference method for assessing water pools, the cost and time involved limit their use in research, clinical, and field settings. Therefore, new methods that assess hydration status safely, accurately, reliably, and feasibly are needed. Bioelectrical Impedance Analysis (BIA) has been used as a simple and feasible method for assessing water pools. However, concerns remain regarding its applicability across MC phases. In summary, evidence is needed regarding variations in strength-related outcomes under different hormonal environments throughout the MC. Additionally, variations in hydration status and water compartments throughout the MC warrant further investigation. Understanding the differences in strength- and hydration-related outcomes between naturally menstruating athletes and those using OCs also requires clarification. Concerns regarding the applicability of BIA during the MC persist, and further clarification is needed to distinguish physiological from bioelectrical characteristics as markers of fluid-related shifts. Considering the gaps mentioned above, the primary aim of this study is to analyse variations in strength-related outcomes (i.e., endurance, maximal, and explosive strength) resulting from hormonal fluctuations across three distinct and identified MC phases (i.e., early follicular, late follicular, and mid-luteal) in naturally menstruating athletes. Secondary aims include: (1) comparing hydration status, energy balance measures (energy expenditure and body composition), and strength-related outcomes (maximal, endurance, and explosive) within and between naturally menstruating athletes and those using OCs; (2) testing the reliability of methods used to assess fluid changes, energy expenditure, and body composition throughout the MC; (3) exploring whether energy availability and resting energy expenditure are associated with sex hormone production across MC phases in naturally menstruating athletes; and (4) testing, validating, and proposing recommendations for the use of bioelectrical impedance in tracking fluid changes throughout the MC.

STUDY DESIGN:

This study will use a longitudinal observational design involving two groups of female athletes: naturally MC athletes and OC users. Participants will be assessed across three distinct phases of their menstrual cycle or oral contraceptive cycle. Testing procedures will be standardized across all sessions to minimize between-session variability. A block randomization approach will be used to determine whether participants will begin testing during the follicular or luteal phase, while OC users will begin assessments either during the withdrawal phase or the active pill phase. For naturally menstruating athletes, day 1 of a new menstrual cycle will be defined as the onset of menstruation. For OC users, day 1 will correspond to the first day of active pill consumption within the 21-day active pill cycle. The identified menstrual cycle phases for naturally menstruating participants will include the early follicular, late follicular, and mid-luteal phases. For OC users, assessments will be conducted during week 1, weeks 2-3, and week 4 of the contraceptive cycle. All assessments will take place at the Exercise and Health Laboratory, Faculty of Human Kinetics, University of Lisbon.

SUBJECTS:

Female athletes (N=40) will participate, divided into two groups: natural MC (N=24) and OC users (N=16).

MEASURES:

Measurements will be conducted across three menstrual cycle phases (i.e., early follicular, late follicular, and mid-luteal) and across the oral contraceptive cycle during week 1, weeks 2-3, and the withdrawal week (week 4). Assessments will include: (i) maximal voluntary isometric strength assessed using handgrip dynamometry, bench press, and leg press; (ii) serum estrogen and progesterone concentrations; (iii) body water compartments and water turnover assessed using dilution techniques; (iv) hydration status assessed using plasma osmolality, sodium concentration, and vasopressin; (v) energy balance assessed using doubly labeled water, and body composition changes assessed using a 4-compartment model; and (vi) resting energy expenditure assessed by indirect calorimetry.

SAMPLE POWER ANALYSIS:

Sample size was calculated based on observed effect-sizes of 0.7 and 0.9, respectively for within (MC phases) and between-group differences (natural MC vs. OC). To detect differences using a type I error of 5% and a power of 80% (G*Power v.3.1.9.2), within two MC phases using an effect size of 0.7, resulting in 15 participants while for detecting differences between groups, 16 athletes would be required. Sixteen athletes per group are needed but 8 additional athletes will be enrolled in the natural MC as ~50% of the exercising women had ovulatory cycles, totalling 40 athletes (24 for the natural MC and 16 under OC).

STATISTICAL ANALYSIS:

Statistical analysis will be performed using IBM SPSS statistics version 28.0 (IBM, USA) and RStudio (Version 1.4.1717, RStudio Team, MA). To perform the block randomization of the testing session to start in either the follicular or luteal phases or to start in the placebo pill or OC pill phase, a computer algorithm written in RStudio (Version 1.4.1717, RStudio Team, Boston, MA) will be employed with randomly selected block sizes. To assess the variation in primary outcomes due to the MC phases (i.e., early follicular, late follicular, and mid-luteal), linear mixed-effects models including the menstrual phases and potential confounding factors will be used. The covariance matrix for repeated measures within subjects over time will be modeled as Unstructured or, if necessary, Compound Symmetry. Model residual distributions will be examined graphically to identify which specific phases differ from each other. To compare natural MC and OC users for the secondary outcomes, mixed-design analysis of variance (ANOVA), with factors (phases) and group (natural MC and OC users), will be performed, and Student's unpaired or paired t-tests will be used post hoc to investigate any significant model effects or interactions. Multiple regression analysis will be employed to test the association between energy availability and sex hormones in natural MC athletes, adjusting for potential confounding factors. To test the validity of alternative techniques for assessing energy expenditure, body composition, and water compartments, Student unpaired or paired t-tests will be performed. Also, multiple regression analysis will be used to investigate the association between alternative and reference methods whereas the agreement between methods will be assessed by using the Bland-Altman approach (also used for testing the reliability of the methods), including the analysis of the correlation between the mean and the difference of the methods. Statistical significance will be set at p<0.05 (2-tailed).

Inclusion Criteria:

• Cisgender female individuals (biologically female, not undergoing gender-transition therapy)
• Age between 18 and 30 years;
• Classified as at least Tier 2 athletes;
• Minimum of 1 year of federated sports participation;
• Menarche occurred at least 3 years prior to enrollment;

Additional inclusion criteria for the natural menstrual cycle group:

• Menstrual cycle length between 21 and 35 days with at least nine consecutive cycles in the previous year;
• Confirmed ovulatory cycle (luteinizing hormone surge and progesterone >16 nmol/L);

Additional inclusion criteria for the oral contraceptive group:

• Use of combined monophasic oral contraceptives with a 21-day regimen;
• Use of the same oral contraceptive for at least 3 months prior to enrollment;

Exclusion Criteria:

• Pregnancy or child birth within the previous 12 months;
• Active smoking;
• Diagnosis of metabolic, cardiovascular, or respiratory disease:
• Use of continuous or extended-cycle oral contraceptives.

Cisgender female individuals (biologically born female individuals not currently under gender-transition therapy)

The study population will consist of 40 female athletes aged 18 to 30 years. Participants will be recruited through Portuguese sports federations and the Portuguese Commission of Olympic Athletes. All participants will be classified as at least Tier 2 athletes with a minimum of one year of federated sports participation. Participants will be allocated into two groups according to menstrual and hormonal status:i) Natural menstrual cycle group (n = 24); ii) Oral contraceptive users group (n = 16). All participants will be cisgender women, biologically female, and not undergoing gender-transition therapy.