Fifty-three healthy eumenorrheic females with moderate physical activity and no history of lower limb disorder were enrolled in the study. Women with moderate physical activity were defined as those who exercised less than seven hours per week and were not participating in competitive-level sports. We had to exclude a total of 31 women: two due to lack of adherence to the protocol, five due to sensitivity to electrical stimulation, three due to the absence of estradiol and/or progesterone fluctuations across the menstrual cycle, eight withdrew from the study, and 13 decided not to participate in any part of the study after being enrolled. Thus, the results presented here are based on data acquired from 22 women (age: 27.0 ± 4.4 years; BMI: 24.1 ± 3.5 kg/m²; cycle length: 30.4 ± 3.9 days; mean ± SD). All participants provided written informed consent. This study was approved by the University of Texas Southwestern Medical Center and the Northwestern University institutional review boards.

Subjects participated in testing sessions every other day for one menstrual cycle. The first testing session was scheduled based on the subject’s preference, resulting in random starting days within the menstrual cycle across subjects. Testing sessions were conducted at a consistent time within the day to minimize the potential effect of diurnal fluctuation of hormone levels across visits per subject (Bao et al., 2003; Bungum et al., 2011). Participants were instructed to maintain stable exposure to caffeine, alcohol, and exercise for 12 h before each testing session.

Each participant was placed in a prone position on a padded examination table with their knee fully extended and their ankle joint secured in a boot. The boot was affixed to a six degrees-of-freedom load cell (Figure 1). The ankle-boot-load cell complex was adjusted such that the lateral malleolus was aligned with the rotational axis of the load cell. The subject position was recorded and used in subsequent testing sessions. Subjects were asked to remain silent and motionless but were instructed to stay awake throughout the testing session.

Venipuncture was performed on the antecubital area at each testing session using a 5 mL Vacutainer serum separator tube (BD, Franklin Lakes, New Jersey). Once the tube was filled, the sample was mixed well and allowed to clot. The samples obtained at Northwestern were processed by Northwestern Memorial Hospital Outpatient Laboratory and the samples obtained at UT Southwestern were sent to MedFusion. The blood samples were used to determine the serum concentrations of estradiol and progesterone. The cyclic patterns of these hormones over time were characterized using a non-linear mixed effect model with harmonics terms (Figure 2). The time was normalized such that the first day of menses was at time 0 and the end of the cycle was at time 1.0. Peak estradiol levels were centered at time 0.5 and the day of peak progesterone level was centered at time 0.75. This model offers an approach to investigating patterns accounting for within and across subject variations over time (Albert and Hunsberger, 2005; Mumford et al., 2012).

Surface electromyography (sEMG) was performed using bipolar electrodes placed on the muscle bellies of the soleus (SOL) and the peroneus longus (PL). Two types of electrodes were used depending on the subject, either pre-amplified surface electrodes (DE-2.1 Bagnoli single differential, interelectrode distance: 10 mm, contact dimensions 10 × 1 mm) or bipolar Ag/AgCl pre-gelled electrodes (diameter 10 mm, inter-electrode distance 2 cm). The sEMG electrode type used in a given subject was consistent throughout the testing sessions. Before electrode placement, the skin was shaved, lightly abraded using Nuprep® (Weaver and Company, CO, United States), and cleansed with water to remove any residue. Each electrode site was measured with respect to bony landmarks and marked with a permanent marker to ensure the exact electrode placement at subsequent testing sessions. Subjects were encouraged not to wash off the markings between sessions. The ankle torque was measured using a six degrees-of-freedom load cell (JR3®, CA, United States) attached to a boot and concentric with the axis of the ankle. Both the EMG and load cell data were amplified (Micro1401 Data Acquisition Unit, Cambridge Electronic Design, United Kingdom) and sampled at 2,000 Hz.

The motor response (M-wave) and H-reflex of SOL muscle were evoked by delivering a 1-ms square pulse from a DS7A constant current stimulator (Digitimer Ltd., Herfordshire, England). The location of the bipolar stimulating electrode (electrode diameter 0.8 cm, inter-electrode distance 2 cm) on the tibial nerve was optimized by moving the stimulating electrode over the nerve until a low-current stimulus elicited the H-reflex. The electrode placement was fixed with a medical tape after a minimal response was observed in the PL muscle while significant SOL muscle reflex response was recorded. The recording started by progressively increasing the stimulus intensity in 1-mA increments starting from 1 mA and continued until the peak-to-peak M-wave had reached a plateau over at least five trials (Figure 3B). The stimuli were delivered at 10- to 20-s random intervals to prevent post-activation depression (Hultborn et al., 1996). The stimulus intensity that generated the maximal peak-to-peak M-wave was considered the maximum stimulus intensity (S-100). Five measurements of the maximum M-wave at 10–20 s intervals were obtained. To compute the associated changes in the contractile properties of the muscle, five stimuli at supramaximal intensity (S-120: 120% of stimulus intensity needed to evoke maximal peak-to-peak M wave) were delivered to the tibial nerve at 10–20 s intervals (Grosset et al., 2005).

The torque data obtained from the load cell were processed offline using Matlab software (R2019a, MathWorks Inc., MA, United States). A fourth-order zero-lag Butterworth low pass filter with a final cut-off frequency of 10 Hz was applied to the torque signal.

Peak torque (PT), time to peak torque (TPT), and half relaxation time (HRT) were measured from the twitch evoked at the maximum stimulus intensity (S-100) and the supramaximal intensity (S-120) (Figure 4). TPT was calculated as the time from the onset of ankle torque to the PT. HRT was defined as the time needed for the muscle to decrease its force to 50% of its maximum.

The twitch response was analyzed at 1) the follicular phase where the estradiol level increased from a low level to its maximum while the progesterone levels remained low, and 2) the luteal phase where the progesterone increased from a low level to its maximum along with corresponding changes in the background estradiol levels.

Data are presented as mean ± SD. The mean values of PT, TPT, and HRT obtained from each subject were used for statistical analyses. The statistical analysis for this study was performed in the R language (version 4.2.2). A mixed-effect model two-way ANOVA was used to analyze the effect of menstrual phase and stimulus intensity on the peak torque (PT) of the muscle. To evaluate the effect of changes in hormone concentrations in the follicular and luteal phases on TPT and HRT, a generalized estimating equation (GEE) was employed using the geepack R package (Ballinger, 2004; Halekoh et al., 2006). Significance was accepted for p values <0.05. A GEE approach is typically used to develop population average models because it take into account the correlation of within subject data which may violate the independence assumptions made by traditional regression procedure (Hubbard et al., 2010).

The estradiol and progesterone profiles obtained from a mixed-effect model with harmonic terms are shown in Figure 2. The cycle day was normalized to the cycle length and centered such that 0 corresponds to the first day of menses, 0.5 corresponds to peak estradiol, 0.75 corresponds to peak progesterone, and 1.0 corresponds to the last day of the cycle. Estradiol concentrations on the first day of menses, peri-ovulatory (peak estradiol concentration), and mid-luteal (peak progesterone concentration) were 44.7 ± 15.4 pg/ml, 315.2 ± 92.4 pg/ml, and 190.3 ± 55.9 pg/ml, respectively. The corresponding progesterone concentrations at those time points were 1.1 ± 0.6 ng/ml, 0.5 ± 0.4 ng/ml, and 12.8 ± 4.3 ng/ml. These concentrations are consistent within the range of estradiol and progesterone concentrations reported in the literature (Sehested, 2000; Marsh et al., 2011; Mumford et al., 2012).

Figure 5 shows the torque profile of the muscle twitch elicited at S-100 of 22 subjects across the menstrual cycle. Analysis of variance of PT within one session is shown in Figure 6. PT collected in two consecutive sessions during menses, at equivalent endocrine states, shows no significant differences (Table 1).

To evaluate the effect of stimulus intensity on the degree of muscle recruitment across the menstrual cycle, the PT obtained at three key time points (menses, peak estradiol, and peak progesterone) were compared. The values did not differ between the sessions at menses, peak estradiol level (peak E), and peak progesterone level (peak P) (Figure 7). There was no significant difference between PT elicited at S-100 and S-120, suggesting that most of the available motor units were recruited at the S-100 stimulus intensity and the aggregate recruitment of these motor units did not change the net force production across the different visits.

The increase in estradiol concentration in the follicular phase of the menstrual cycle (normalized day 0 to day 0.5) was not associated with either TPT or HRT (Table 2, p > 0.05). In the luteal phase (normalized day 0.5 to day 1), TPT was not associated with the fluctuation of either estradiol or progesterone. As indicated in Table 2, GEE revealed a main effect of progesterone (b = −0.007, p-value = 0.006) and estradiol × progesterone interaction (b = 4.7 × 10⁻⁵, p-value = 0.002) on HRT. Our data suggest that an increase in progesterone in the luteal phase predicts decreases in HRT better than chance depending on the concentration of estradiol.

Evidence in the literature regarding estradiol-mediated changes in muscle twitch characteristics in humans is scarce. Sarwar and colleagues found that the half relaxation time of the quadriceps muscle increased by up to 20% at the peri-ovulatory phase compared to menses, while Janse De Jonge and Kubo demonstrated that muscle twitch characteristics across the menstrual cycle were invariant (Sarwar et al., 1996; Janse De Jonge et al., 2001; Kubo et al., 2009). These conflicting results may be attributed to the low-resolution testing over the menstrual cycle and the potential differences in the relative levels of progesterone and estradiol during the luteal phase between the two studies. We argue that our every other day study design has a higher likelihood to capture the relationship between sex hormones and muscle twitch characteristics.

We found that an increase in estradiol over a short period (10–14 days exposure) expressed no association with soleus muscle peak force, and activation and de-activation dynamics. These findings are consistent with results from previous studies of the soleus muscle (Kubo et al., 2009) but not the quadriceps muscle (Sarwar et al., 1996). One distinguishing feature between the soleus and the quadriceps muscle is in their fiber composition. The soleus is primarily composed of type I fibers while the quadriceps muscles have a higher component of type II fibers (Edgerton et al., 1975; Staron et al., 2000). In rats, it has been suggested that estradiol may exert a differential effect on muscles with different fiber compositions due to a higher estrogen receptor (ER) content in type I fibers (Lemoine et al., 2002; Brown et al., 2009). While it is unlikely that the discrepancy in relaxation times between Sarwar et al., Janse De Jonge et al., Kubo et al., and our study are related to estradiol’s classical genomic pathway, it is still plausible that estradiol could have a differential effect on type II fibers via an ER-independent mechanism. While the exact mechanism is unknown, it could be suggested that estradiol targets the calcium handling of the fiber via the sarcoplasmic reticulum, which is the primary driver of the relaxation of the muscle (Harridge et al., 1996), and may explain the difference in relaxation times over the menstrual cycle between the quadriceps muscles and the soleus.

In our paradigm, during the activation phase of the twitch, the observed torque at the joint level is an aggregate effect of the muscle and the tendon. Tendon, a collagenous tissue, is affected by the change in estradiol concentrations in animal models (Lee et al., 2004), but similar data in humans is limited. Several examinations on the effect of changing estradiol on the laxity of similar collagenous connective tissue have been reported in humans. For example, Casey et al. reported that the anterior knee laxity in eumenorrheic women was not significantly different throughout the menstrual cycle, suggesting that collagen-based connective tissue mechanics were not modulated with changes in systemic levels of estradiol (Casey et al., 2014). Burgess et al. observed that the length, cross-sectional area, and stiffness of the Achilles tendon did not change throughout the menstrual cycle (Burgess et al., 2009). On the other hand, several other studies have shown that anterior knee laxity was modulated at different phases of the menstrual cycle (Shultz et al., 2004; Zazulak et al., 2006). Taken together, these findings suggest that the inconsistency in the literature on the effect of estradiol on musculotendon twitch properties may have origins in both the tendon and muscle components.

The impact of progesterone on muscle twitch characteristics remains inconclusive. In agreement with Kubo et al., 2009, our data indicated that during the luteal phase, fluctuation of estradiol concomitant with progesterone (in a physiologically relevant state) was not associated with any change in the muscle peak torque, time to peak torque, and half relaxation time (GEE regression parameters was close to zero). These findings are in direct contrast with the prevailing hypothesis that progesterone, through its vascular effects (White et al., 1995), may alter muscle force-generating capacity (Yasuda et al., 2009). Further studies examining the effect of isolated progesterone treatment, as well as combined estrogen-progesterone treatment, on muscle force-generating capacity are warranted.

We hypothesized that following progesterone treatment, HRT would shorten. In humans, high-dose progesterone (100–200 mg/day for 10–20 weeks) has been used as a treatment to prevent pre-term labor (Norwitz and Caughey, 2011) by inducing muscle relaxation through the production of nitric oxide (Renzo et al., 2005). It is important to note that during these interventions, recipients additionally express systemically high estradiol levels due to pregnancy (Soldin et al., 2005). To what extent the clinically observed effect of progesterone in this cohort is mediated or synergistically influenced by the background estradiol levels remains unknown. Our data indicate that during the normal co-fluctuation of the two hormones in the luteal phase, the effect of progesterone seems to be dependent on the estradiol concentration; a finding that highlights the complex interactions between the two hormones and warrants further examination.

In recent years, there has been increasing interest in tailoring the female athletic training regime around their menstrual cycle (Bruinvels et al., 2022) to optimize the athlete’s performance. We argue that the menstrual cycle-specific phase training paradigm is premature given the conflicting evidence linking cycle state and injury (McNulty et al., 2020; Nédélec et al., 2021). Evidence exists that the risk of joint injury is greater in the late follicular phase, a state of increased concentration of estradiol. Contrary to what we expected, our results demonstrated that the acute change in estradiol concentration during ovulation does not affect muscle twitch properties, a basic unit in muscle recruitment and function at the joint level. This inconsistency suggests that the increase in the frequency of injury around ovulation may not be attributed to the effect of estradiol on muscle but may be attributed to the estradiol-modulated effect on the neural state (Smith and Woolley, 2004). One can also observe that the reduction in the rate of injury in the second half of the cycle cannot exclude the potential effect of sex hormones. Our data did show that changes in half-relaxation time were associated with the combined change in progesterone and estradiol in the luteal phase. However, the hormone-associated changes may be minimal.

One could argue that an unaccustomed bout of exercise or stimulation can result in muscle damage, and this damage could alter the muscle twitch properties, thus, confounding our results. In the literature, it has been shown that electrical stimulation of muscle can significantly increase serum creatine kinase concentrations, an indirect measure of muscle damage (Aldayel et al., 2010; Vanderthommen et al., 2012). However, these protocols utilized 5 to 6-second-long contractions at 40%–100% of the maximum force. In this study, there were a total of 5 stimuli, each electrical stimulus has 1 ms duration, and the interstimulus interval was 10–15 s. For each stimulus, the twitch contraction period was about 200 ms (Figure 5). Our within session data showed an insignificant difference between the first and the fifth stimuli with the largest change of PT within subjects being 1.25 Nm. Thus, there was no within session electrical stimulation induced fatigue. In addition, changes in ankle torque during maximal voluntary contraction across all visits were also not significant. Taken together, these findings suggest the absence of potential neuromuscular adaptations or muscle damage within and across visits.

The present study investigated the possible association between the fluctuation of estradiol and progesterone and muscle twitch characteristics. The data we collected and our ability to interpret the findings are not without limitations, including a small sample size. An additional uncontrolled factor is a possibility that muscle twitch characteristics are also dependent on a complex interaction with other hormones, such as testosterone (Prezant et al., 1993; Hirschberg et al., 2020) and relaxin (Dehghan et al., 2014), factors that were not incorporated in the present design. Future investigations should also include the measurement of muscle damage-associated biomarkers such as creatine kinase to help monitor any potential muscle damage that may be caused by an unaccustomed bout of exercise or stimulation.

This is the first study to use an every other day testing paradigm to investigate the sex hormone-mediated change of muscle contraction properties. This testing paradigm allowed for the analysis of the effect of changes in estradiol levels and the combined effect of progesterone and estradiol levels on musculotendinous properties. Further exploration is necessary to unfold the mechanism of interaction between elevated concentrations of estradiol and progesterone and muscle activation and deactivation dynamics.