# Passive hamstring muscles rigidity throughout the menstrual cycle and the effect of oral contraception

Jérémie Bouvier, Mélanie Prudent, Emeric Stauffer, Maxime Pingon, Alexandre Fouré, Cyril Martin

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Jérémie Bouvier, Mélanie Prudent, Emeric Stauffer, Maxime Pingon, Alexandre Fouré, et al. Passive hamstring muscles rigidity throughout the menstrual cycle and the effect of oral contraception. Journal of Science and Medicine in Sport, 2025, 10.1016/j.jsams.2025.01.008. hal-04951498v2

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# Passive hamstring muscles rigidity throughout the menstrual cycle and the effect of oral contraception

Jérémie Bouvier<sup>a</sup>, Mélanie Prudent<sup>b</sup>, Emeric Stauffer<sup>a,b</sup>, Maxime Pingon<sup>b</sup>, Alexandre Fouré<sup>a,1</sup>, Cyril Martin<sup>a,*,1</sup>

Keywords:

Contraceptive pill

Shear wave elastography

Passive muscle mechanical properties

Musculoarticular stiffness

Hormones

## ABSTRACT

**Objectives:** Female athletes have a lower risk of hamstring strain injuries than males. The variations in oestradiol and progesterone concentrations happening during the menstrual cycle and oral contraceptive use may influence muscle mechanical properties, potentially affecting muscle injury risk. However, to date, no study has combined an assessment of hamstring muscles' mechanical properties spread over the full knee range of motion with rigorous hormonal control of the menstrual cycle, electromyographic monitoring and inclusion of oral contraceptive users. The present study aims to be the first to rigorously evaluate the association between menstrual cycle, oral contraceptive use and hamstring muscles' mechanical properties.

**Design:** Cross-sectional study with repeated measures.

**Methods:** Shear wave elastography measurements were performed at three different times during the menstrual and oral contraceptive cycle. Hormonal assessment and electromyographic monitoring were also carried out.

**Results:** No difference in hamstring muscles' shear wave speed, anatomical cross-sectional area, knee joint musculoarticular stiffness and maximal range of motion were observed along the menstrual cycle despite the presence of the expected variations in ovarian steroid hormones concentrations. No difference in these parameters was also reported throughout the oral contraception use.

**Conclusions:** No differences in the mechanical properties of the hamstring muscles were observed, whether the concentrations of ovarian steroid hormones fluctuate with the menstrual cycle or remain stable with oral contraceptive use, despite the notable effects of these hormones on the muscle composition.

## Practical implications

* It is not necessary to consider the phase of the menstrual cycle when assessing passive hamstring muscle rigidity, knee range of motion, or musculoarticular stiffness, with the hip flexed.

* The different stages of oral contraception use can also be overlooked whilst assessing these parameters.

* The fluctuations in ovarian steroid hormones occurring throughout the menstrual cycle do not seem to affect the anatomical cross-sectional area of the hamstring muscles.

## 1. Introduction

A growing body of evidence reports different rates of muscle injury depending on the phase of the menstrual cycle (MC), suggesting a possible role for hormonal fluctuations in the risk of such injuries.<fnref n="1" />⁻³ Although one study found no differences in muscle injury rates across the MC,<fnref n="1" /> another reported rates to be 3, 5, and 6 times greater during the late follicular, luteal, and premenstrual phases compared to menstruation,² whilst a third one found rates to be twice as high during the late follicular phase as during menstruation, with no significant differences between the luteal phase and menstruation.³ These findings suggest that the sex hormones 17β-oestradiol (E2), and possibly progesterone (P4), whose concentrations vary throughout the MC, may have a detrimental effect on the risk of muscle injury. Indeed, human muscle tissue expresses receptors for both of these hormones,⁴ which, upon ligand binding, translocate to the cell nucleus to act as transcription factors for target genes.⁵,⁶ Moreover, E2 and P4 have been shown to differently alter the composition of the human extracellular matrix.⁷,⁸ Specifically, E2 decreases the amount of type I collagen in connective

<footnote><sup>a</sup> Université Claude Bernard Lyon 1, LIBM, Laboratoire Interuniversitaire de Biologie de la Motricité, UR 7424, UFR STAPS de Lyon, F-69622 Villeurbanne, France</footnote>

<footnote><sup>b</sup> Explorations Fonctionnelles Respiratoires, Médecine du sport et de l'Activité Physique, Hospices Civils de Lyon, Hôpital Croix Rousse, Lyon, France</footnote>

<footnote>Abbreviations: E2, 17β-oestradiol; EF, early follicular phase; LF, late follicular phase; MC, menstrual cycle; ML, mid luteal phase; OC, oral contraception; P4, progesterone; SWS, shear wave speed.</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: cyril.martin@univ-lyon1.fr (C. Martin).</footnote>

<footnote n="1">¹ Alexandre Fouré and Cyril Martin contributed equally.</footnote>
tissue cell cultures,⁷,⁸ whereas P4 promotes fibroblast proliferation and increases the amount of type I procollagen.⁷ As a change in the composition of muscle passive structures may contribute to the onset of a muscle injury,⁹,¹⁰ it seems conceivable that the fluctuations in E2 and P4 concentrations occurring throughout the MC may be involved in the processes underlying muscle injury.¹¹ Furthermore, the use of monophasic low-dose oral contraceptives (OCs), which drastically attenuates these variations in E2 and P4 concentrations, is widespread in the sporting context and differences in extracellular matrix metabolism¹² and in susceptibility to muscle damage¹¹ have been reported between OC users and non-users. Indeed, the muscle collagen fractional synthesis rate is elevated in response to exercise in eumenorrheic women but not in OC users,¹² who also experience slower recovery from post-exercise strength loss and increased perceived muscle soreness.¹¹

Hamstring strains are among the most prevalent injuries in female sports, such as football¹³ and athletics.¹⁴ As muscle injury mechanisms are partly dependant on the muscle strain,¹⁵ the potential association of the MC with hamstring's response to stretch has been investigated using shear wave elastography measurements. Shear wave elastography is considered as the reference non-invasive method to assess localised muscle mechanical properties.¹⁶ Recent shear wave elastography studies did not demonstrate any modulation of muscle rigidity throughout the MC.¹⁷⁻¹⁹ However, only one of these scarce studies included rigorous hormonal control of the MC¹⁸ and none of them included OC users. Yet, blood sampling is mandatory to ensure the occurrence of the hormonal variations inherent to a physiological MC, especially in exercising females.²⁰ The inclusion of OC users may also be of interest, as OCs down-regulate the MC-associated variations in E2 and P4. This both ensures that no confounding factors alter the parameters measured in eumenorrheic females independently of their fluctuations in E2 and P4 concentrations and that a significant proportion of the exercising female population is not overlooked. Furthermore, the shear wave elastography measurements from these previous studies were not systematically distributed over large ranges of motion, whereas these measurements are influenced by the muscle lengths over which they are performed,²¹ emphasising the importance of characterising muscle properties over full ranges of motion. These measures are also highly sensitive to minimal muscle activity,²² but none of the aforementioned studies performed an electromyographic monitoring to ensure the passive state of the muscles.

The aim of the present study was therefore to determine whether the mechanical properties of the hamstring muscles change throughout the MC or with OC intake in females, using a rigorous hormonal control of the MC. We hypothesised that hamstring muscle rigidity would be lower during the MC phases associated with high E2 and P4 concentrations in the MC group, and that these variations would not occur in the OC group.

## 2. Methods

### 2.1. Participants

Forty active females were included in this study according to their menstrual status: 26 were eumenorrheic whilst 14 took OCs (Fig. 1). Eumenorrheic females presented regular MCs during the three months preceding their inclusion, a MC being considered as “regular” if its length did not differ by more than four days from the length of the previous one. The length of these three MCs had to be comprised between 21 and 35 days and all participants were free from any medication that could affect the hormonal variations bound to a regular MC for at least six months.²³ The participants from the OC group were all taking 2nd generation low-dose monophasic ethinylestradiol/progestin OCs for at least six months prior to inclusion. They were all aged between 18 and 35 years old, did not present any cardiac symptoms and had not suffered a hamstring injury in the six months preceding the experiment, nor any other musculoarticular disorder that would have prevented

<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Inclusion flow diagram.</figcaption></figure>

Five MC participants experienced a slightly longer MC than expected, which resulted in the exclusion of a single testing session for each of them based on hormonal measurements (2 late follicular and 3 mid luteal sessions in total). MC: menstrual cycle, OC: oral contraception.

them from freely practising sport in the two years prior to enrolment. They were all involved in some form of sport before and during the experiment, had no muscle pain or illness and were not using any form of medication that could have influenced the parameters measured in the study. Exclusion criteria comprised pregnancy, previous anterior cruciate ligament reconstruction and an E2 concentration less than 30 % higher in LF and ML than in EF combined with a P4 concentration less than 30 % higher in ML than in LF (i.e., corresponding to a value higher than the maximal inter-assay variation coefficient of the assays performed in this study).

All participants volunteered to be part of the protocol and provided written informed consent before their participation. This study was conducted in accordance with the latest version of the Declaration of Helsinki, was approved by a relevant independent ethics committee (Comité de Protection des Personnes Sud-Est IV, Centre Léon Bérard, on 07/27/2022, ID-RCB: 2022-A00806-37) and was registered on ClinicalTrials.gov (NCT05802277).

An *a priori* power analysis showed that a group of 20 participants would allow the detection of a medium effect size from Cohen's *d* assessment (i.e., corresponding to a Hedge's *g* = 0.6 or to a change in mean of 13 %) in SWS between menstrual cycle phases with an *α* equal to 0.05 and a sufficient power (1 - *β* = 0.80). This sample of 26 MC participants was intended to preserve statistical power despite potential exclusions associated with MC disturbances.

### 2.2. Study design

Each participant took part in three identical testing sessions conducted in a randomised order during the menstrual cycle or OC use. The three testing sessions were scheduled at the same time of day and
consisted of a blood sample, a physical activity questionnaire (administered only during the first session), anthropometric measurements, an assessment of maximal knee joint range of motion, musculoarticular stiffness, surface electromyographic activity, anatomical muscle cross-sectional area, shear wave elastography, and maximal voluntary contraction torque (Fig. 1). Participants were asked to avoid any strenuous exercise or hamstring stretching for 48 h before each session.

## 2.3. Testing sessions

### 2.3.1. Menstrual cycle monitoring

A combination of a calendar-based approach with ovarian hormonal blood sampling was employed to control for the participant's MC length and regularity. For the MC group, starting days of three to six past MCs (i.e., menses' first day) were gathered to compute an average MC duration. Based on this calculation, the testing sessions were scheduled according to a block randomisation during the next expected early follicular (EF) phase (between the 1st and 4th days of the following cycle), late follicular (LF) phase (14 or 15 days before the first day of the next predicted cycle) and mid luteal (ML) phase (between 6 and 8 days before the first day of the next predicted cycle). Testing sessions for the OC group were conducted on equivalent days of the OC cycle (i.e., from the 21st to the 24th pill for phase 1 [placebo pills], from the 6th to the 7th and from the 13th to the 15th for phases 2 and 3 respectively [both active pills]). Participants could not be blinded either, as they were already tracking their menstrual or OC cycle before their participation in the study. At the beginning of each session, a 3 mL blood sample was drawn from an antecubital vein into a plain tube without gel. It was immediately refrigerated at 4 °C for 3.8 ± 1.7 h before being centrifuged and frozen at -80 °C. 17β-oestradiol and progesterone serum concentrations were measured in duplicate with enzyme-linked immunosorbent assays following the manufacturer's instructions (Cayman Chemical Company, MI, US).

### 2.3.2. Questionnaire and anthropometric data

Participants were asked to complete the global physical activity questionnaire. Lower leg length was determined by measuring the distance from the lateral malleolus to tibial condyle and thigh length was defined as the distance between the lateral distal femoral epicondyle and greater trochanter.

### 2.3.3. Maximal knee range of motion

The participants were asked to lay on their back with the right knee joint flexed at 90° and the hip flexed at 70° (180° representing a complete extension). Their pelvis and right ankle were firmly strapped to the isokinetic dynamometer (CON-TREX MJ, Physiomed, Germany). A support was positioned against the anterior surface of the thigh to stabilise it throughout the knee extensions. Maximal knee range of motion (RoMmax) was determined as the maximal tolerable stretching pain during a manual slow passive knee extension at 5°/s or when achieving a complete knee extension (i.e., 185°) and the best of two attempts was recorded. All passive procedures described from this point onwards were conducted under constant electromyographic monitoring (as detailed below).

### 2.3.4. Musculoarticular stiffness

The knee joint was then passively extended for five conditioning extension-flexion cycles at an angular velocity of 5°/s from 90° of knee flexion to 80% of the participants' RoMmax.³⁸ The knee angular position and knee joint torque signals were acquired at 2 kHz via a Powerlab system (16/35, ADinstruments, Oxford, UK) using the Labchart software (v.8.1.21, ADinstruments, Oxford, UK). These signals were low-pass filtered with a 10 Hz cut-off frequency and torque was corrected for gravity. A second-order polynomial model was fitted to the torque-position relationships, which enabled the calculation of

musculoarticular stiffness for a knee angle of 120° (i.e., the largest common angle among all participants) as follows:

$$ \text{Stiffness} (\theta) = 2a\theta + b $$

where a and b are experimental constants and θ is knee position.²⁴ The knee joint torque acquired during passive knee extensions was also determined for each knee angle relative to RoMmax and stiffness was assessed for 80% RoMmax.

### 2.3.5. Ultrasonography

A line was drawn at 50% of the thigh length to guide the ultrasound explorations. The boundaries of the biceps femoris long head (BF), semitendinosus (ST) and semimembranosus (SM) were first located with B-mode ultrasound transverse imaging using an Aixplorer ultrasound device (Mach30, v.2.1.0.3395, Supersonic Imagine, Aix-en-Provence, France) coupled with a linear transducer array (5–18 MHz, SuperLinear 18-5, Vermon, Tours, France) and marked on the skin. Ultrasonographic images were acquired along this line in transverse planes to evaluate the hamstring muscles' cross-sectional area. The muscle anatomic cross-sectional area was measured by manually outlining the muscle shape with ImageJ (ImageJ 1.53t, NIH, Bethesda, MD, USA).

### 2.3.6. Shear wave elastography

Next, shear wave elastography maps were acquired as videos during the extension phase of knee extension-flexion cycles at 2°/s (Fig. 2A). These maps were spread over 1 cm² and obtained at a frequency ranging between 1.2 and 2.0 Hz depending on their depth (range: 1.2–3.7 cm). The ultrasound device's persistence was set on "medium", smoothing level on "5" and optimisation on "penetration". A pressure sensor (Flexiforce A401, Barneveld, Netherlands) was placed on the "record" button of the ultrasound device to synchronise the shear wave elastography measurements with the knee joint torque and angle via the

<figure><img src="image_3.png" /><figcaption>**Fig. 2. A** Typical shear wave elastography maps obtained for the biceps femoris long head (BF), semitendinosus (ST) and semimembranosus (SM) during passive knee extension and shear wave speeds measured along B the menstrual cycle and C oral contraception use at a knee angle of 120°.</figcaption></figure>

The images in A depict maps of shear wave elastography superimposed on the corresponding ultrasound image for different knee angles, including 120°, the angle at which the measurements for graphs B and C were taken, n = 20 MC and 12 OC. EF: early follicular phase, LF: late follicular phase, ML: mid luteal phase.
Powerlab system. Shear wave elastography recordings were exported and processed with the Matlab software (R2018b, The MathWorks, Natick, USA) using the ElastoGUI toolbox (https://bio.tools/elastogui). Shear wave speed (SWS, index of rigidity) was quantified in the whole regions of interest for every frame of each shear wave elastography video. The corresponding knee angle was determined for each SWS measurement to evaluate SWS-angle relationships for each muscle from 90° to 120°. This process was repeated to assess SWS-angle relationships from 90° of knee angle to 80 % of each participant's RoMmax. The SWS at 120° of knee angle and 80 % RoMmax was linearly interpolated and considered for linear mixed-effect analyses. The complete joint amplitude (i.e., every 1 % of RoMmax) was used for statistical parametric mapping (SPM) evaluations.

### 2.3.7. Surface electromyographic activity

Before the assessment of RoMmax, the skin was shaved and cleaned with alcohol, and pairs of Ag/AgCl 40 × 36 mm surface electrodes (Medico Electrodes, Uttar Pradesh, India) were positioned as close as possible to the shear wave elastography measurement sites on each hamstring muscle. Raw surface electromyographic signals were acquired at 2 kHz via the Powerlab system and an amplifier (FE234 Quad BioAmp, ADinstruments, Oxford, UK) and were notch (50 Hz) and band-pass (20–400 Hz) filtered. The root mean square was calculated on a 150 ms sliding time window and normalised to the maximal value reached during isometric maximal voluntary contractions (as detailed below). The average root mean square of surface electromyographic signals was computed for each extension–flexion cycle. During the whole experimental sessions, participants could then monitor the electromyographic activity of their hamstring muscles through visual real-time feedback. The investigator also carefully checked for the absence of any low-level muscle contraction that might have raised the electromyographic signals above their baseline, to ensure the resting state of BF, ST and SM.

### 2.3.8. Isometric maximal voluntary contractions

With the right knee joint flexed at 90° and the hip at 70°, participants were asked to warm up their knee flexors by performing several submaximal isometric knee flexions. After a two-minute recovery period, two isometric maximal voluntary contractions were performed, separated by a one-minute rest. The best of two attempts was recorded to normalise the electromyographic signals to their maximum value.

### 2.4. Reproducibility assessment

Measurements of anatomical cross-sectional area, RoMmax, passive musculoarticular stiffness and passive muscle SWS were realised during two identical sessions separated by 48 h according to the procedure described above. The reproducibility of these measures was quantified in 12 participants (6 females [age: 23.5 ± 1.5 years, height: 1.70 ± 0.04 m, mass: 58 ± 6 kg] and 6 males [age: 25.3 ± 5.7 years, height: 1.76 ± 0.08 m, mass: 63 ± 19 kg]) with intraclass correlation coefficient (ICC(3,1)), standard error of measurement (SEM) and coefficient of variation (CV).

### 2.5. Statistics

Statistical analyses were performed using R (version 4.1.1., The R Foundation for Statistical Computing). Student's t-test was used to evaluate between-group differences in anthropometric measurements and global physical activity questionnaire results. Linear mixed-effect analyses were conducted using the lme4 R package with intercepts for participants as random effects. Visual inspection of residual plots revealed no deviations from homoscedasticity or normality, nor any violation of linearity assumption. Absence of collinearity and of influential data points was carefully checked. Bonferroni correction for multiple comparisons was applied to the post-hoc analyses and associated effect sizes (ESs) were computed using Hedge's g.

Blood E2 and P4 concentrations were analysed with *group* and *phase* as fixed effects. Analyses of muscle cross-sectional areas were performed between *phases* in separate models for each *group* and *muscle*, and between *groups* in separate models for each *muscle*. RoMmax, musculoarticular stiffness and maximal voluntary torque values were analysed between *phases* for each *group* and between *groups* (all *phases* averaged). Interactions of *phase* and *muscle* factors for SWS analyses were evaluated in both *groups* separately. Between-groups comparisons were conducted with *muscle* as fixed effect (all *phases* averaged). Regarding the SWS analyses, only the results involving a *group* or *phase* effect were covered here. Significant *group* or *phase* effects were only presented if they interacted with *muscle* factor to avoid grouping SWS values from different muscles.

SPM was applied to evaluate the influence of *phase* and *muscle* on these SWS data over the entire range of absolute knee angles reached by each participant (i.e., from 0% [90°] to 100% of the maximal absolute knee angle reached during the passive stretch). Two-way repeated measures analysis of variance (SPM was performed for each group using the spm1d MATLAB package.²⁵ In cases of non-significant interaction, one-way repeated measures analyses of variance (SPM were conducted to test the *phase* main effect on SWS values for each muscle and each group. Data are presented as mean ± standard deviation and the significance level was set at p < 0.05.

Considering only the participants who reached their RoMmax before attaining a full knee extension, the data processing and statistical analyses described above were conducted for knee angles relative to each participant's RoMmax (from 0% [90°] to 80%) (Supplementary material 1).

## 3. Results

### 3.1. Reproducibility assessment

The measurements of the passive hamstring muscles' SWS, cross-sectional area, RoMmax and musculoarticular stiffness showed a very good reproducibility (all ICC > 0.80). The ICC, SEM and CV computed for each parameter are displayed in Table S1. Table S2 displays the reproducibility of the SWS and musculoarticular stiffness measured for knee angles relative to each participant's RoMmax.

### 3.2. Questionnaire and anthropometric data

No between-groups difference was observed in global physical activity questionnaire results and anthropometric measurements (p > 0.05, Table 1). The average MC length in the MC group was 30.4 ± 2.0 days. The mean testing days for each cycle phase for both groups are displayed in Table 1.

### 3.3. Blood samples

A significant *phase* × *group* interaction was observed for E2 concentration (F = 9.14, p < 0.001). In the MC group, it was greater during the LF phase than during the EF (p < 0.001, ES = 1.65, large effect) and ML ones (p = 0.020, ES = 0.62, medium effect, Fig. 3A). It was also greater during the ML phase than during the EF phase (p = 0.005, ES = 0.98, large effect, Fig. 3A). E2 concentration did not differ across phases in the OC group (p = 1.00). There was no group-difference between the EF phase and phase 1 (p = 1.00) whilst E2 concentration was greater for the MC group during the LF phase than phase 2 and during the ML phase than phase 3 (both p < 0.001, ES = 1.94 and 1.52, large effects).

A significant *phase* × *group* interaction was observed for P4 concentration (F = 25.33, p < 0.001). In the MC group, it was greater during the ML phase than during the EF and LF phases (both p < 0.001, ES = 2.50 and 2.32, large effects, Fig. 3B). The concentrations in P4 during
<table><caption>**Table 1**

Participants' characteristics, global physical activity questionnaire scores and testing days according to both menstrual cycle (MC) and oral contraceptive (OC) groups.</caption><thead><tr><th></th><th>MC group</th><th>OC group</th><th>p-Value<br>Group<br>effect</th></tr></thead><tbody><tr><td colspan="4"><strong>Anthropometric measurements</strong></td></tr><tr><td>Age (years)</td><td>22.4 ± 3.4</td><td>21.0 ± 2.0</td><td>0.159</td></tr><tr><td>Height (m)</td><td>1.65 ± 0.08</td><td>1.66 ± 0.07</td><td>0.811</td></tr><tr><td>Body mass (kg)</td><td>60 ± 8</td><td>58 ± 7</td><td>0.405</td></tr><tr><td>Thigh length (cm)</td><td>40.9 ± 3.7</td><td>41.8 ± 2.9</td><td>0.635</td></tr><tr><td>Leg length (cm)</td><td>34.6 ± 2.8</td><td>35.0 ± 1.5</td><td>0.471</td></tr><tr><td colspan="4"><strong>Global physical activity questionnaire results</strong></td></tr><tr><td>Weekly physical activity<br>(MET-min/week)</td><td>6048 ± 3750</td><td>5638 ± 1977</td><td>0.689</td></tr><tr><td>Daily lying and sitting time (min)</td><td>440 ± 172</td><td>340 ± 129</td><td>0.073</td></tr><tr><td colspan="4"><strong>Testing days (days)</strong></td></tr><tr><td>EF phase/phase 1</td><td>2.8 ± 1.9</td><td>22.4 ± 1.2</td><td>-</td></tr><tr><td>LF phase/phase 2</td><td>16.2 ± 4.1</td><td>7.3 ± 0.8</td><td>-</td></tr><tr><td>ML phase/phase 3</td><td>22.4 ± 2.5</td><td>14.7 ± 1.1</td><td>-</td></tr></tbody></table>

The reason why the test days for the MC and OC groups do not align is that the EF phase is traditionally associated with the pill-free days, which begin after taking the 21st pill. MET: metabolic equivalent of task, EF: early follicular, LF: late follicular, ML: mid luteal.

the ML phase for the MC group were greater than those observed in phase 3 for the OC group (p < 0.001, ES = 2.88, large effect, Fig. 3B).

### 3.4. Maximal knee range of motion

No significant *phase* effect was observed in MC (F = 1.55, p = 0.226, ES = 0.14, negligible effect) and OC (F = 2.69, p = 0.090, ES = 0.17, negligible effect) groups. No *group* effect was reported (F = 1.72, p = 0.199, ES = 0.48, small effect Table 2). These analyses yielded similar results when considering only the participants who reached their RoMmax before attaining a full knee extension (Supporting information 1, Table S3).

### 3.5. Anatomical cross-sectional area

No significant *phase* effect was observed on muscle anatomical cross-sectional area in both MC (BF: F = 2.71, p = 0.081, ES = 0.27, small effect|ST: F = 1.64, p = 0.208, ES = 0.26, small effect|SM: F = 0.37, p = 0.691, ES = 0.06, negligible effect) and OC (BF: F = 0.47, p = 0.630, ES = 0.14, negligible effect|ST: F = 2.55, p = 0.103, ES = 0.28, small effect|SM: F = 0.01, p = 0.993, ES = 0.01, negligible effect) groups. No significant difference between *groups* was reported for the three hamstring muscles (BF: F = 0.34, p = 0.564, ES = 0.19, small effect|ST: F = 0.28, p = 0.604, ES = 0.16, small effect|SM: F = 0.02, p = 0.892, ES = 0.06, negligible effect, Table 2).

### 3.6. Torque-angle relationship

Model adjustments to experimental torque-angle curves were excellent (mean R² = 0.997 ± 0.005 [range: 0.952-1.000]). No main effect of *phase* was observed on musculoarticular stiffness in the MC group (F = 0.70, p = 0.506, ES = 0.17, negligible effect) and OC group (F = 0.54, p = 0.593, ES = 0.11, negligible effect, Table 2, Fig. 4). No significant difference between *groups* was either observed (F = 2.59, p = 0.118, ES = 0.63, medium effect, Fig. 4). These analyses yielded similar results for knee angles relative to each participant's RoMmax considering only those who reached it before attaining a full knee extension (Supporting information 1, Table S3 and Fig. S1).

### 3.7. Shear wave speed

No significant *phase* * muscle interaction was observed in the MC group (F = 0.74, p = 0.567) and OC group (F = 0.25, p = 0.908) (Fig. 2B and C). Regarding between-groups differences in SWS, no significant *group* * muscle interaction was reported (F = 1.37, p = 0.256). The SPM analyses showed no interaction between *phase* and *muscle* and no *phase* main effect across the entire range of absolute knee angles investigated in both *groups* (p > 0.05, Figs. S2 and S3). These analyses yielded similar

<figure><img src="image_4.png" /><figcaption>**Fig. 3.** A Blood 17β-oestradiol (E2) and B progesterone (P4) concentrations throughout menstrual and oral contraception cycles.</figcaption></figure>

Significant differences are represented as * for p < 0.05, ** for p < 0.01 and *** for p < 0.001 with horizontal brackets for *cycle phase* effects in MC group and vertical ones for between *groups* differences, n = 20 MC and 12 OC. EF: early follicular phase, LF: late follicular phase, ML: mid luteal phase, MC: menstrual cycle, OC: oral contraception.

<figure><img src="image_5.png" /></figure>
<caption>**Table 2**</caption>

Descriptive values of maximal knee extension range of motion (RoM<sub>max</sub>), maximal voluntary contraction torque, surface electromyographic signals, anatomical cross-sectional area and musculoarticular stiffness for the three cycle phases in the menstrual cycle (MC) and oral contraception (OC) groups.

<table><thead><tr><th rowspan="2"></th><th colspan="3">MC group</th><th colspan="3">OC group</th></tr><tr><th>EF phase</th><th>LF phase</th><th>ML phase</th><th>Phase 1</th><th>Phase 2</th><th>Phase 3</th></tr></thead><tbody><tr><td>RoM<sub>max</sub> (°)</td><td>75.2 ± 12.9</td><td>73.8 ± 14.8</td><td>71.3 ± 13.5</td><td>77.9 ± 13.2</td><td>80.6 ± 13.9</td><td>81.4 ± 12.7</td></tr><tr><td>Maximal voluntary contraction torque (N·m)</td><td>86.2 ± 17.3</td><td>86.5 ± 17.6</td><td>83.3 ± 17.4</td><td>85.6 ± 11.4</td><td>88.2 ± 12.8</td><td>88.1 ± 12.3</td></tr><tr><td>Surface electromyographic signals (%)</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Biceps femoris long head</td><td>2.1 ± 0.9</td><td>2.1 ± 0.8</td><td>2.4 ± 0.9</td><td>2.5 ± 1.3</td><td>2.3 ± 1.0</td><td>2.5 ± 1.1</td></tr><tr><td>Semitendinosus</td><td>2.2 ± 1.3</td><td>2.0 ± 0.8</td><td>1.9 ± 0.9</td><td>2.5 ± 1.3</td><td>2.6 ± 1.4</td><td>2.4 ± 1.2</td></tr><tr><td>Semimembranosus</td><td>2.4 ± 0.8</td><td>2.7 ± 1.0</td><td>2.8 ± 1.2</td><td>3.5 ± 1.7</td><td>2.7 ± 0.9</td><td>2.9 ± 1.0</td></tr><tr><td>Anatomical cross-sectional area (cm²)</td><td>22.0 ± 2.8</td><td>22.7 ± 3.1</td><td>22.0 ± 3.4</td><td>22.3 ± 4.0</td><td>22.9 ± 4.3</td><td>22.8 ± 3.8</td></tr><tr><td>Biceps femoris long head</td><td>8.9 ± 1.6</td><td>9.0 ± 1.9</td><td>8.3 ± 2.0</td><td>9.0 ± 2.1</td><td>9.4 ± 2.6</td><td>8.9 ± 2.1</td></tr><tr><td>Semitendinosus</td><td>4.6 ± 1.1</td><td>4.9 ± 1.3</td><td>5.1 ± 1.1</td><td>4.8 ± 1.5</td><td>5.0 ± 1.4</td><td>5.4 ± 1.2</td></tr><tr><td>Semimembranosus</td><td>8.5 ± 2.2</td><td>8.7 ± 2.4</td><td>8.6 ± 2.3</td><td>8.5 ± 2.3</td><td>8.5 ± 1.7</td><td>8.5 ± 1.9</td></tr><tr><td>Stiffness (N·m·s⁻¹)</td><td>0.27 ± 0.10</td><td>0.29 ± 0.12</td><td>0.29 ± 0.11</td><td>0.20 ± 0.10</td><td>0.22 ± 0.12</td><td>0.22 ± 0.11</td></tr></tbody></table>

EF: early follicular, LF: late follicular, ML: mid luteal.

results for knee angles relative to each participant's RoM<sub>max</sub> considering only those who reached it before attaining a full knee extension (Supporting information 1, Figs. S4, S5 and S6).

### 3.8. Maximal voluntary isometric contraction

No phase effect was observed for the maximal torque for any group (MC: F = 0.86, p = 0.433, ES = 0.12, negligible effect | OC: F = 0.66, p = 0.525, ES = 0.13, negligible effect) and no effect of group was reported (F = 0.15, p = 0.702, ES = 0.12, negligible effect, Table 2).

## 4. Discussion

The objective of the present study was to determine whether the mechanical properties of females' hamstring muscles evolve throughout the MC or with OC intake. We report the first elastography assessments of the hamstring muscles performed over the full knee range of motion during multiple phases of the MC and OC use, in conjunction with electromyographic and hormonal controls. In contrast to our initial hypothesis, the main results showed that the SWS of all hamstring muscles, knee joint stiffness and RoM<sub>max</sub> remained stable along the MC. As expected, similar results were observed for the OC group.

The surges in E2 and P4 concentrations observed in the MC group during LF and ML phases were consistent with the pattern of hormonal changes that would be expected for regular MCs. Additionally, the concentrations observed in the OC group were in accordance with a regular OC use.<sup>26</sup>

The endogenous variations in E2 and P4 concentrations reported in the present study did not translate into any modifications of passive hamstrings' mechanical properties, knee joint passive stiffness or RoM<sub>max</sub> throughout the MC. However, endogenous variations of E2 and P4 concentrations may affect muscle composition. For example, in a cell culture of human fascia lata fibroblasts, type-I collagen production decreased when E2 concentration was raised for 48 h to a level similar to that of the LF.<sup>4</sup> Moreover, exposure of two cell cultures of human ligaments to E2 and P4 concentrations mirroring those found during MCs during 24 h demonstrated a dose-dependent reduction in fibroblast proliferation and type-I procollagen quantity with increasing E2 concentration.<sup>8</sup> As a significant proportion of the whole muscle passive tension is borne by its extracellular matrix,<sup>27,28</sup> changes in collagen metabolism can result in modification of passive muscle mechanical properties along the menstrual cycle. Nevertheless, no changes in the parameters assessed in the present study were observed. Similar results have previously been reported for shear

<figure><img src="image_6.png" /><figcaption>**Fig. 4.** A Torque-angle relationships measured during passive knee extension and B the associated musculoarticular stiffness for each group and cycle phase. Standard deviations are not shown in A for clarity and musculoarticular stiffness is calculated for a knee angle of 120° in B, n = 20 MC and 12 OC. EF: early follicular phase, LF: late follicular phase, ML: mid luteal phase, MC: menstrual cycle, OC: oral contraception.</figcaption></figure>
wave elastography measurements¹⁷ and knee joint RoMmax.²⁹ However, the sole two studies that assessed hamstring musculoarticular stiffness at different times during the MC, using either knee³⁰ or hip³¹ stretches, reported contrasting results. One of them showed a lower knee joint stiffness during the LF phase whilst the other one reported no MC-associated changes in hip joint stiffness.³⁰,³¹ Considering that none of these studies demonstrated changes in endogenous E2 and P4 concentrations between MC phases, the present study is the first to report hamstring musculoarticular stiffness values in such a reliable way, showing its stability throughout the MC.

One might legitimately think that analysing SWS from a single knee angle could also have clouded some MC-associated adaptations, especially as muscle length is the main determinant of a muscle's passive SWS. However, multiplying the levels of this factor in our linear mixed-effect analyses would have dramatically affected their statistical power, whilst SPM has been designed to analyse continuous data. The SPM results displayed in Figs. S2 and S3 demonstrate that the F-values never reached their significance threshold, indicating that the SWS was not affected by the MC phase in any hamstring muscle for any knee angle.

All SWS and knee joint stiffness analyses were conducted at the same absolute angles for every participant although they had different RoMmax [45.8–94.1°]. However, it could be considered that a determined joint angle may not correspond to the same location on the passive tension–length relationship of participants with different RoMmax. Hence, the aforementioned statistical analyses were also conducted for knee angles relative to each participant's RoMmax. Considering only the participants who reached their RoMmax before achieving the full knee extension, SWS, musculoarticular stiffness and RoMmax were not different according to menstrual status (Table S3, Figs. S1, S4, S5 and S6).

In the present study, elastography measurements were performed in a single 1 cm² area in the middle of each hamstring muscle. However, muscle passive mechanical property adaptations seem to be site-specific.³² The localised assessment of mechanical properties conducted in the present study could thus have prevented the observation of adaptations occurring throughout the MC. Moreover, the tendon bears most of the passive increase in the muscle–tendon complex length at the beginning of the stretch.³³ Its mechanical properties could be altered throughout the MC considering the effects of E2 and P4 on collagenous tissues.⁷,⁸,³⁴ However, knee musculoarticular stiffness, which provides an indication of the global stiffness of all the structures crossing this joint, did not evolve between MC phases in the present study. Thus, even if a local change in the mechanical properties of the hamstrings occurred, the global musculoarticular stiffness was not significantly affected by the MC or the OC intake.

Differences in musculoarticular stiffness between sexes can be abolished when normalised to muscle volume, suggesting that passive torque production is dependent on muscle size.³⁵ The stiffness stability observed throughout the MC in the present study could thus have been confounded by potential changes in muscle volume occurring during the MC. Indeed, the drop in ovarian hormone concentrations caused by the ovariectomy of female rats led to a reduction of their muscle fibres' cross-sectional area,³⁶,³⁷ whereas E2 supplementation resulted in an increase in this parameter.³⁷ These considerations formed the rationale for assessing muscle cross-sectional area during the different phases of the MC in the present study, revealing no relationship between this parameter and the endogenous variations of ovarian hormones.

## 5. Conclusion

The evaluation of hamstring passive mechanical properties using elastography over a restricted common or a large knee joint range of motion revealed no link with the hormonal variations associated with either the menstrual cycle or oral contraception. In addition, stable

knee joint stiffness, RoMmax and hamstring muscles' cross-sectional areas were observed along the MC and OC use in the present study. Further research is needed to determine the hamstring muscles' rigidity during voluntary contraction throughout the menstrual cycle and with oral contraceptive use.

## Funding information

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

## Confirmation of ethical compliance

The study was approved by a relevant independent ethics committee (Comité de Protection des Personnes Sud-Est IV, Centre Léon Bérard, on 07/27/2022, ID-RCB: 2022-A00806-37) and was registered on [ClinicalTrials.gov (NCT05802277).](https://ClinicalTrials.gov)

## Declaration of interest statement

The authors declare no known financial or personal conflict of interest that could have influenced the work reported in this paper. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

## Acknowledgements

We extend our sincere gratitude to professors D. Roussel and V. Pialoux and to the whole department of functional respiratory testing of the Croix-Rousse hospital for their invaluable assistance in the handling of our blood samples. We would also like to thank all participants for their unwavering commitment throughout the multiple testing sessions, which greatly contributed to the proper advancement of this study.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at [https://doi.org/10.1016/j.jsams.2025.01.008](https://doi.org/10.1016/j.jsams.2025.01.008).

## Data availability

All data acquired in the present study are available from the corresponding author.

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