Effect of menstrual cycle and contraceptive pill phase on aspects of exercise physiology and athletic performance in female athletes: protocol for the Feminae international multisite innovative project

Sample size

Each site will aim to recruit and test a minimum of 10 EUM and 10 COCP users, resulting in an expected total of 70 EUM participants and 70 COCP users in the final combined dataset. The group size was based on a balance between pragmatic considerations of the recruitment capacity across the different sites and a range of simulations of mixed effects models with random effects (participants and sites) and small overall effect sizes (eg, hedges g of 0.1–0.2) likely to generate adequate power (1-beta~0.8).

EUM group

Participants will: (1) have MC lengths ≥ 21 days and ≤ 35 days resulting in nine or more consecutive periods per year; (2) provide evidence of a luteinising hormone surge; (3) demonstrate the required hormonal profile as defined by Elliott-Sale et al3 ; and (4) have not used any type of hormonal contraceptive for a minimum of 3 months, but ideally 6 months, before enrolment. MC phase tracking (figure 1) will be achieved by (1) denoting the first and last day of menstruation on a calendar for each cycle; (2) undertaking urinary ovulation detection using kits (visual confirmation will be provided to the researcher); and (3) providing a phase 4 blood sample. A posteriori exclusion will be applied to participants not meeting the stipulated hormonal profiles. Two of the ‘tracking phase’ visits will be used as familiarisation sessions or two separate familiarisation sessions will be scheduled at times convenient for the participants, in addition to the mandated tracking phases session where blood samples need to be provided. EUM participants will be tested during phases 1, 2 and 4 of their cycle.

Combined, monophasic contraceptive pill (COCP) group

Participants must have been taking their COCP ≥ 3 months before recruitment and must be taking a combined, monophasic, second-generation COCP (see table 2 for permitted brands).

Phase tracking (figure 1) will be achieved by (1) denoting the first and last pill-taking days on a calendar and (2) providing a pill-taking (days 18–20) and pill-free (days 5–7) blood sample. A posteriori exclusion will be applied to data from testing time points that do not comply with the stipulated hormonal profiles. Two of the ‘tracking phase’ visits will be used as familiarisation sessions. COCP users will be tested once during the pill-taking phase (any day between days 14 and 21) and once (days 4, 5, 6, or 7) during the pill-free phase.

Venous blood sample

A trained phlebotomist will draw a blood sample from an antecubital vein. Approximately 10 mL will be collected into a serum tube. Serum samples will be left to clot for 30 min before being centrifuged at room temperature (~20°C). Samples will be centrifuged at 3000 g for 10 min and then stored in a −80°C freezer for the subsequent determination of endogenous 17 β-oestradiol and progesterone. Samples will be transported between laboratories on −20°C dry-ice or in a −80°C freezer and will only be subjected to one freeze–thaw cycle.

Height and body mass

Using a stadiometer, height will be measured in bare feet, with minimal clothing and no impeding hairstyles. Participants will be positioned in the ‘Frankfort Plane’ and asked to take a deep breath, which they hold, while the measurement is taken. Height will be measured at the first laboratory visit only. Body mass will be measured at every testing session by an experimenter using the same calibrated scales on each occasion (ie, within and between participants), and the participants will wear minimal clothing and no shoes.

Standardised warm-up and stretching

Participants will complete a 5 min, 15–100 W (self-selected, but recorded and replicated for each subsequent testing session) warm-up on a cycle ergometer. Participants will then undertake the following warm-up stretching exercises, which will be led/supervised by an experimenter: modified Hurdler’s stretch, kneeling stretch, supine hamstrings stretch, overhead triceps stretch, seated bent-knee biceps stretch and child’s pose.

Balance: stork balance stand test

In bare feet, participants will place their hands on their hips and then position their non-supporting foot against the inside knee of the supporting leg. They will be given 1 min to practice the balance. They must raise their heel to balance on the ball of the foot. The stopwatch will be started as the heel is raised from the floor. The stopwatch will be stopped if any of the following occurs: (1) the hand(s) come off the hips; (2) the supporting foot swivels or moves (hops) in any direction; (3) the non-supporting foot loses contact with the knee; (4) the heel of the supporting foot touches the floor. The total time in seconds will be recorded. One minute’s rest will be allowed between attempts. Three attempts using the same supporting leg will be offered. However, if improvements are made, the participants may continue to perform the test beyond three attempts. The outcome measure is the longest total time of a single test recorded in seconds. Bouteraa et al10 reported an intraclass correlation coefficient (ICC) of 0.90 for repeated stork static balance test trials in female basketball players.

Muscular endurance: push-ups

The push-up test will be administered with participants starting in a modified knee push-up position (legs together, lower leg in contact with the mat, back straight, hands shoulder-width apart, head up, using the knees as the pivotal point). The participants will raise their body by straightening their elbows and returning to the ‘down’ position until their chin or chest touches the mat. Their stomach should not touch the mat. The participant’s back must remain straight at all times, and the participant must push up to a straight arm position. The test will be stopped when the participant strains forcibly or cannot maintain the appropriate technique within two repetitions. The outcome measure is the maximum number of push-ups performed consecutively without rest. An ICC of 0.83 was previously reported in college-age women performing bent-knee push-ups.11

Agility: Illinois Agility Run test

The length of the course will be 10 m, and the width will be 5 m. Four cones will be used to mark the start, finish and the two turning points. Another four cones will be placed down the centre an equal distance apart. Each cone in the centre will be spaced 3.3 m apart. The participant will be asked to lie on their front (head to the start line) with their hands by their shoulders. On the ‘Go’ command, the stopwatch will be started, and the participant must get up as quickly as possible and run around the course in the direction indicated, without knocking the cones over (or stepping over the cones), to the finish line, at which point the timing will be stopped. The total time in seconds will be recorded. The score will be the best of three attempts, and 1 min rest will be allowed between attempts. If improvements are made, participants may continue to perform the test beyond three attempts. Kutlu et al12 reported an ICC of 0.98 for repeated Illinois Agility Run test trials in amateur female football players. However, it should be noted that Kutlu et al12 used timing gates, a standing start and a minimum of 3 min rest between trials.

Muscular strength: handgrip

The participant will hold the dynamometer in their dominant hand, with their arm at a right angle, wrist slightly extending, and elbow by their side. The dynamometer handle will be adjusted if required—the base should rest on the first metacarpal (heel of palm), while the handle should rest on the middle of the four fingers. When ready, the participant will squeeze the dynamometer with maximum isometric effort, which will be maintained for about 5 s. No other body movement will be allowed. The participant will be strongly encouraged to give a maximum effort. The score is the best of three attempts; 1 min’s rest will be permitted between attempts. If improvements are made, participants may continue to perform the test beyond three attempts. Using an identical protocol, Gerodimos et al13 showed good reliability in 90 male basketball players, with an intraclass correlation (2,1) of 0.99 and a standard measurement error of 1.24 kg.

Power: countermovement jump

This test will be administered using the MyJump2 app, which calculates jump height by recording the jump and accurately selecting its take-off and landing. A smartphone or tablet will be positioned at the same height (1 m height) and the same distance (1.5 m) from the participant, enabling a clear view of the participant’s lower limbs. The sagittal plane is used to identify the exact take-off and landing frames more easily. Arm-swing will be prohibited; participants must keep their hands on the hips throughout the test. The test administrator will pay strict attention to the participant’s hands to ensure they are not used to apply additional force through the legs. Verbal instructions will be given to participants to rapidly descend by flexing at the knees and hips to approximately 90° and then immediately extend their knees and hips to ‘jump’ vertically up off the ground as high as possible, landing back on the mat on both feet at the same time. A 1-min rest will be allowed between trials. The take-off will occur from both feet simultaneously, with no initial steps or shuffling. Participants will not be allowed to pause at the bottom position of the squat. The best result of three attempts will be recorded. However, if improvements are made, participants may continue to jump beyond three attempts. It is also important that the athlete attempts to land in the same position as they took off, as jumping forwards, backwards, or sideways can affect the test results. To aid this, experimenters should stick tape to the floor as a marker for participants to take-off from and land on. The outcome measure is the highest jump recorded. Test-retest reliability of the CMJ using the My Jump app showed an ICC of 0.94 in recreationally active women.14

Flexibility: sit and reach

This test will involve sitting on the floor with legs stretched straight ahead. The soles of the participant’s bare feet will be placed flat against the box. Both knees will be locked and pressed flat to the floor; the tester may assist by holding them down. With the palms facing downwards and the hands on each other or side by side, the participant must reach forward along the measuring line as far as possible. Their hands must remain at the same level, not reaching further forward than the other. After some practice, they must reach out and hold that position for one to two seconds while the distance is recorded. There must not be any jerky movements. The score is the best of three attempts, and 1 min rest is allowed between attempts. If improvements are made, participants may continue to perform the test beyond three attempts. The outcome measure is the furthest score recorded. Jackson and Langford15 showed good reliability across two measurements in adult women (mean 35.1±7.5 cm, r²=0.99) using a similar protocol. The protocol must be standardised, as differences in muscle temperature and stretching exercises between testing phases will alter sit-and-reach distance. It is common for improvements in sit-and-reach scores to improve during the practice trials.

Incremental cycling test

Before and following the cycling protocol, a note will be made of any unique details of the test environment (ie, temperature, humidity, pressure), performance, or athlete concerns in the comments section of the data sheet. The incremental cycling tests will be performed on an electronically braked cycle ergometer, and oxygen uptake (V̇O₂) and carbon dioxide output (V̇CO₂) will be determined using open-circuit spirometry, open circuit spirometry and a breath-by-breath metabolic cart. Baseline heart rate (HR), blood lactate (La−) and blood pressure will be obtained before starting. All blood samples will be taken from the earlobe or finger; however, this must be consistent within a given research site. Participants will complete a 5-min warm-up at 45 W. The consumption of water is permitted during the warm-up.

Part 1: The protocol will encompass approximately five 4-min stages. The starting power output (W) will be amended for different training tiers/performance levels (see examples below). The familiarisation trials will help to determine the starting power output, which will be one of the following 15 W increments (ie, 45, 60, 75, 90, 105 W).

• Example 1: 45—warm-up; stage 1–45; stage 2–60; stage 3–75; stage 4–90; stage 5–105. This may not be enough to induce a blood lactate concentration of 4 mmol/L in some trained participants.

• Example 2: 45—warm-up; stage 1–90; stage 2–105; stage 3–120; stage 4–135; stage 5–150.

Participants can ride at a self-selected cadence, but not below 60 rev/min (recommend between 70 and 100 rev/min) and the cadence can vary up to 10% of the starting cadence during the test if desired. For each stage, power output, average HR, and rating of perceived exertion (RPE (0–10)) in the last 30 s, and blood [La−] values in the last 15 s will be recorded using a standardised spreadsheet. In sites where possible, blood pressure will be recorded for each increment within the last 30 s. Once a blood [La−] of ≥ 4 mmol/L is recorded, the protocol will be stopped, and the participant will receive a 5-min rest; the participant may remove the mouthpiece, and water may be consumed during this time, but they should remain seated on the cycle ergometer without pedalling. The last completed 4 min workload will be recorded as the maximal 4-min power output.

Part 2: the participant will be asked to begin pedalling at their preferred cadence against no load for 1 min. The test will begin at a one-step power output (ie, 15 W) higher than the last completed 4-min power output eliciting ≥4 mmol/L in part 1. After that, the load is increased by 15 W every 30 s until voluntary exhaustion. HR will be recorded at the end of each 30 s increment. Pedal cadence will be maintained within 10% of the participant’s preferred cadence established in part 1, and completion of the incremental cycling test will be signified by volitional cessation of exercise or an inability to maintain a pedal cadence. During the latter part of the test, the experimenter will verbally encourage the participants until voluntary termination. HR, power output, and RPE (RPE (0–10)) will be recorded at exhaustion. Participants will remain seated on the cycle ergometer for 3 min without pedalling, after which a single blood [La−] sample will be taken. Participants will then undertake a cool-down at a self-selected power output for 5 min.

A 7-breath rolling average will process all breath-by-breath files per Robergs and Burnett’s recommendations16 before data are extracted.

Part 1: 30-s averages for V̇O₂ (mL/kg/min and L/min), V̇CO₂, respiratory exchange ratio (RER), and HR will be determined for each 4-min stage. A third-order polynomial regression curve will represent the lactate response to exercise (power output and blood [La−]). These data will be used to determine Dmax, the exercise intensity that yields the maximum perpendicular distance from the third-order polynomial plotted curve from the lactate measures to the straight line between the first and the last data point,17 and onset blood [La−] accumulation at 4 mmol/L (OBLA18). Data will be analysed using The Lactate Thresholds App (ExPhysLab available at https://www.exphyslab.com/lactate/about, supported by R package lactater 0.1.1). Linear regression will then determine HR and V̇O₂ at Dmax and OBLA. The outcome measures will be Dmax power output (W), blood [La−] and corresponding HR, and V̇O₂, and OBLA 4 mmol/L power output, corresponding HR, and V̇O₂.

Part 2: peak power output will be determined as the last stage a participant completes fully. If a participant finishes partway through a 30-s increment, peak power will be calculated as a percentage of the duration they completed the last power output. V̇O_2peak will be determined by averaging data in the last 30 s of the test. To ensure maximum effort, the following variables will also be reported at the end of the test (averaged 30 s RER *(criteria > 1.1), HR as a percentage of age-predicted maximum (Max HR_agepredicted=220 age) (criteria > 95% HRmax), blood [La−] concentration (+3 min (criteria > 7 mmol/L)) and RPE (at exhaustion (criteria > 9/10)). The outcome measures will be V̇O_2peak (mL/kg/min and L/min), maximum HR and RER.

There is limited research in trained populations investigating the reliability of physiological variables obtained during incremental exercise tests comprising 4-min stages. However, from the limited literature available, the reliability of the OBLA is high (r=0.92) in trained women and men using an incremental running protocol comprising of 4-min work stages.19