# Muscle contraction type does not influence the etiology of the immediate crossover fatigue evidenced after submaximal unilateral exercises of knee flexor muscles

Jennifer Gioda, Enzo Piponnier, Florian Monjo, Jonathan Bredin, Baptiste

Corcelle, Flavio da Silva, Serge S Colson

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Jennifer Gioda, Enzo Piponnier, Florian Monjo, Jonathan Bredin, Baptiste Corcelle, et al. Muscle contraction type does not influence the etiology of the immediate crossover fatigue evidenced after submaximal unilateral exercises of knee flexor muscles. Heliyon, 2025, 11 (5), 10.1016/j.heliyon.2025.e42801. hal-04978271

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<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
Research article

Muscle contraction type does not influence the etiology of the
immediate crossover fatigue evidenced after submaximal
unilateral exercises of knee flexor muscles

Jennifer Gioda<sup>a</sup>, Enzo Piponnier<sup>a</sup>, Florian Monjo<sup>a,b</sup>, Jonathan Bredin<sup>a,c</sup>, Baptiste Corcelle<sup>a</sup>, Flavio Da Silva<sup>a,1</sup>, Serge S. Colson<sup>a,1,∗</sup>

ARTICLE INFO

Keywords:

Contralateral

Motor performance fatigue

Perceived motor fatigue

Torque-time integral

A B S T R A C T

Motor performance fatigue can occur in a non-exercised muscle following a unilateral exercise performed with the contralateral homologous muscle. While many studies have investigated this crossover fatigue phenomenon, the influence of muscle contraction type is unsettled. This study examined the occurrence and etiology of crossover fatigue after submaximal isometric (ISO), concentric (CONC) or eccentric (ECC) contractions of the knee flexors (KF). In separate sessions, thirteen participants performed unilateral ISO, CONC or ECC KF exercises inducing a 20 % reduction in maximal voluntary isometric contraction (MVIC) of the exercised limb (EL). Motor performance fatigue (i.e., MVIC, voluntary activation [VA], electrically evoked muscle responses [Pt100Hz and Pt10Hz-to-Pt100Hz ratio]) and perceived muscle soreness (PMS) of both EL and non-exercised limb (NEL) were assessed before (PRE), immediately (POST), and 24 h post-exercise (POST24). Global perceived motor fatigue (PMF) was also measured. At POST, significant reductions in MVIC were observed for both EL (−27.7 %; p < 0.001) and NEL (−8.5 %; p < 0.01) indicating the presence of crossover fatigue. At POST, Pt100Hz (−7.5 %; p < 0.05) was impaired in both limbs whereas VA was altered in the EL only (−2.6 %; p < 0.001). PMF was increased at POST only (p < 0.001). Finally, PMS scores were greater at POST (p < 0.01) and POST24 (p < 0.001) than PRE values regardless of the limb considered. At POST24, MVIC, Pt100Hz, VA and PMF had returned to PRE values. Muscle contraction type did not influence crossover fatigue of the KF nor its etiology.

1. Introduction

After engaging in unilateral muscle exercise, the non-exercised contralateral homologous muscle may experience a decrease in maximal voluntary force production (i.e., defined here as motor performance fatigue) [1]. This phenomenon known as “crossover fatigue” [2–4] remains a topic of ongoing debate. A recent systematic review with meta-analysis suggested that the existence of

<footnote><sup>a</sup> Université Côte d'Azur, LAMHESS, France</footnote>

<footnote>b Université Savoie Mont Blanc, LIBM, Chambéry, France</footnote>

<footnote>c Centre de Santé Institut Rossetti-PEP06, Nice, France</footnote>

<footnote>* Corresponding author.
E-mail address: serge.colson@unice.fr (S.S. Colson).
¹ Co-authorship: contributed equally to the present work.</footnote>
crossover fatigue on the contralateral homologous muscle is trivial [4]. The controversy surrounding crossover fatigue may stem from the significant variability in study designs, including factors such as age, sex, training status, upper body versus lower body muscles exercised, outcome measures, and severity of induced fatigue [4,5]. Although these factors may play a role in crossover fatigue, there is a lack of information regarding the influence of muscle contraction type performed during the unilateral exercise. To the best of our knowledge, only two studies have compared the possible occurrence of crossover fatigue after concentric versus eccentric contractions [6,7]. Unfortunately, none has evidenced crossover fatigue in the non-exercised contralateral homologous muscle. Therefore, the influence of muscle contraction type performed during a unilateral exercise on crossover fatigue has yet to be determined.

Other studies that the above-mentioned have evidenced crossover fatigue immediately after a unilateral exercise [3,8–10]. More precisely, reduced voluntary activation in the non-exercised contralateral homologous muscle was reported after isometric [3,10] or eccentric exercises [8,9], suggesting the involvement of central fatigue. More surprisingly, two studies have observed peripheral fatigue supported by impairments of the potentiated resting twitch of the non-exercised contralateral homologous muscle after isometric exercises [3,11]. For example, a ~20 % decrease of the resting twitch was observed in the non-exercised contralateral first dorsal interosseous muscle [11]. In Post et al. study [11] during the unilateral isometric exercise of the ipsilateral muscle, the electromyographic activity of the non-exercised contralateral first dorsal interosseous muscle progressively increased up to 38 % of its maximal activity, possibly contributing to the 10 % crossover fatigue observed (i.e., decrease in maximal voluntary isometric force production). This associated involuntary contraction in the non-exercised contralateral muscle during ipsilateral muscle exercise [12] has been suggested to participate to the crossover fatigue phenomenon [11]. However, in Doix et al. study [3], although a crossover fatigue of ~10.5 % (i.e., decrease in maximal voluntary isometric force production) was reported, only a ~2 % decrease of the potentiated doublet twitch torque was reported in the non-exercised contralateral knee extensors. In addition, in a recent study, although a ~7 % decrease in maximal voluntary isometric force of the non-exercised plantar flexors was reported, no significant resting single twitch decrease was observed after a unilateral eccentric exercise [9]. Unfortunately, in these later studies [3,9] the electromyographic activity of the non-exercised contralateral muscle was not controlled. Although muscle groups studied and/or experimental procedures could account for these conflicting observations, the influence of muscle contraction type performed during the unilateral exercise on central and peripheral fatigue mechanisms involved in the occurrence of crossover fatigue is unsettled. To date, in crossover fatigue studies, central fatigue was observed in the non-exercised contralateral limb after isometric [3,10] and eccentric [8,9] exercises but inconsistent observations were reported regarding peripheral fatigue for these two contraction types [3,9,11]. In addition, to the best of our knowledge, no study has investigated the effects of a unilateral concentric exercise on central and peripheral fatigue in the non-exercised contralateral limb. In some studies investigating motor performance fatigue in the ipsilateral limb after different muscle contraction type, conflicting results were reported regarding central and peripheral fatigue [13,14]. For example, in a study inducing the same level of motor performance fatigue in the knee extensors after maximal concentric and eccentric exercises, central fatigue occurred after the eccentric exercise only, whereas peripheral fatigue was similar after both types of exercise [13]. In contrary, in the only study comparing maximal isometric, concentric and eccentric fatiguing exercises in the knee extensors, peripheral fatigue was greater after the eccentric exercise, whereas central fatigue was similar after all contraction types inducing comparable motor performance fatigue [14]. Taken together, these conflicting observations emphasize the need to examine the influence of muscle contraction type on both motor performance fatigue and crossover fatigue-induced central and peripheral alterations.

Along with the possible central and/or peripheral fatigue alterations, it has been suggested that an increase in subjective perceived fatigue induced by the unilateral exercise performed (i.e., defined here as perceived motor fatigue) [1] may play a role in the altered performance of the contralateral homologous muscle [4,5]. Perceived motor fatigue comprises discomfort and/or pain perception associated with the exercise [1]. To the best of our knowledge, only two crossover fatigue studies evaluated pain perception through the measurement of perceived muscle soreness after unilateral eccentric exercise of knee extensors [8] and plantar flexors [9]. Increased pain perception of the exercised knee extensor muscles was observed at 24 and 48 h after the exercise [8]. In the exercised plantar flexors, greater pain perception was reported immediately after the exercise, lasting up to 72 h [9]. In these two studies, the authors argued that ascending pathways mediating nociceptive information may have inhibited motor cortex areas related to the contralateral homologous muscle and/or that a global pain-induced alteration in cerebral motor planning led to the decreased motor performance of the non-exercised muscle. Interestingly, and although non-exercised, pain perception was also reported (i.e., immediately and lasting until 72 h) in the contralateral homologous muscle after the exercise of the ipsilateral plantar flexors [9]. Although pain perception associated with the exercise could participate to the crossover fatigue phenomenon, the influence of muscle contraction type on perceived motor fatigue in crossover fatigue studies is unknown.

Despite recent advancements in understanding crossover fatigue, more research is necessary to explore the influence of muscle contraction type. In light of this knowledge gap, the present investigation aimed to compare the effects of unilateral submaximal isometric (ISO), concentric (CONC), and eccentric (ECC) exercises of the knee flexor muscles on the occurrence of crossover fatigue and its underlying mechanisms. Based on the current conflicting literature, it is extremely challenging to formulate a sound hypothesis regarding the influence of muscle contraction type on crossover fatigue. However, considering that a similar motor performance fatigue will be induced in the exercised limb (EL), we hypothesized that crossover fatigue in the non-exercised limb (NEL) would similarly occur immediately after ISO, CONC and ECC exercises. We assumed that both central and peripheral fatigue alterations and increased perceived motor fatigue (i.e., encompassing perceived muscle soreness) would contribute to the crossover fatigue in the NEL.
## 2. Materials and methods

### 2.1. Participants

Sample size was determined a priori using G*Power (version 3.1.9.6; Kiel University, Kiel, Germany) based on a recent study evidencing crossover fatigue after a submaximal eccentric exercise performed at 80 % of the maximal voluntary contraction [9] like the present investigation. The minimum sample size obtained was ten participants in a repeated-measure, within-between interaction analyses (power set at 0.95) with two times of measure. Thirteen healthy young males regularly involved in recreational sport activities [mean ± standard deviation (SD); age: 22.6 ± 2.85; body mass: 79 ± 10.63 kg; height: 180.5 ± 4.23 cm] took part in this investigation. The power analysis sensitivity (α = 0.05, power = 0.80) computed with the participants' sample size enrolled in the present investigation led to a large effect size of f = 0.43. Participants were free from lower limbs injuries and did not experience lower limbs surgery (i.e., at least for six months). The experiments as well as risks, discomforts and benefits of the investigation were explained to each participant. An individual written informed consent was obtained before the start of the experiments. Experimental procedures were conducted in accordance with the Declaration of Helsinki latest version (2013). The investigation was approved by an Ethics Committee (Protection Committee of People for Biomedical Research Southeast III, France; Authorization Number 2020-A02811-38).

### 2.2. Experimental design

Throughout a 6-week period, participants were required to visit the laboratory on 9 occasions (Fig. 1): three times for each muscle contraction type (ISO, CONC and ECC). Each muscle contraction type assessment followed the same experimental design including: i) a familiarization session (visit 1), ii) a testing session comprising measurements taken before (PRE) and immediately after (POST) a unilateral fatiguing exercise (visit 2), and iii) a testing session conducted 24 h after the unilateral fatiguing exercise (POST24; visit 3). ECC was always assessed first while ISO and CONC testing were randomized (Fig. 1). A minimum interval of one week was set between each muscle contraction type testing.

During the familiarization sessions, participants were accustomed with the materials and the experimental procedures used to assess knee flexors (KF) motor performance in both the right EL and the left NEL (see details below). During, CONC and ECC familiarizations, participants were acquainted with the angular velocity to maintain during the right EL knee flexions and extensions (i.e., 10°.s⁻¹). Once familiarized with all procedures, the 1 Repetition Maximum concentric or eccentric contraction (1RM CONC and 1RM ECC, respectively) of the EL was measured by adding loads (e.g., progressively reduced from 5 kg to 1 kg) until the participant could

<figure><img src="image_2.png" /><figcaption>Fig. 1. Experimental procedure.</figcaption></figure>

After an eccentric (ECC) familiarization session, participants realized the ECC testing session including measurements in the right EL (exercised limb) and the left NEL (non-exercised limb) realized before (PRE); immediately after (POST) and 24 h after (POST24) the unilateral submaximal exercise performed at 80 % of 1 Repetition Maximum eccentric contraction (1RM ECC). Then, in a random order following a respective familiarization session, participants carried out a concentric (CONC) at 80 % of 1 Repetition Maximum concentric contraction (1RM CONC) or an isometric (ISO) testing session at 70 % of MVIC. Pt₁₀₀Hz and Pt₁₀Hz: potentiated paired twitch torques electrically evoked at 100Hz and 10Hz, respectively.
not maintain the targeted angular velocity. Trials were interspaced by 90s rest periods. In these dynamic familiarization sessions, participants were also accustomed with the unilateral fatiguing exercise to perform (i.e., 5 submaximal ISO or CONC or ECC contractions) in the right EL. In the ISO familiarization session, maximum voluntary isometric contraction (MVIC) of the KF was determined to familiarize the participants with the unilateral submaximal fatiguing isometric exercise. These familiarization procedures allowed for a quick determination of 1RM CONC, 1RM ECC or MVIC during the respective testing session.

At the beginning of the testing sessions (i.e., PRE, POST24), participants rated their perceived muscle soreness on each lower limb and their perceived motor fatigue foreach muscle contraction type (i.e., ISO, CONC, ECC). Then, a specific warm-up comprising “Extender” and “Diver” exercises [15] and single-leg bridges movements performed (two sets of six repetitions for each exercise in the EL and NEL). During the “Extender”, the lower limb was maintained in an extended position for 5s followed by a 5-s rest and before reaching the extended position again. During the “Diver”, the position was reached over a 3-s period followed by a 1-s return. For single-leg bridges, the participants had 1s to reach the extension position, which was maintained for 2s and followed by 1s to return to the starting position. After this, the participants were installed on the ergometer to complete the warm-up by performing submaximal isometric contractions in the testing position. The intensity (in the percentage of their perceived MVIC) of these contractions inter-spaced by 30-s rest periods was progressively increased from ~30 % (4 contractions) ~50 % (3 contractions), ~70 % (2 contractions) to ~90 % (1 contraction). Once the warm-up ended, KF motor performance was assessed with the right EL always tested first, followed by the motor performance measurements in the left NEL. KF motor performance comprised two MVICs superimposed with a paired electrical stimulation (100 Hz). Potentiated stimulations were delivered at 2 s (100 Hz paired stimulus), 4 s (10 Hz paired stimulus) and 6 s (1 Hz single stimulus) after each MVIC. Following this, the 1RM CONC or 1RM ECC of the right EL was determined in the CONC and ECC testing session, respectively. After the unilateral fatiguing exercises (POST), only one MVIC associated with superimposed and potentiated stimulations was performed in the EL and NEL. Then, the participants had to rate their perceived muscle soreness in the EL and NEL and their perceived motor fatigue (Fig. 1).

## 2.3. Unilateral fatiguing exercises

Depending on the fatiguing exercise implemented in the right EL, the exercise intensity was fixed at 70 % of the MVIC for ISO, at 80 % of the 1RM CONC for CONC [14] and at 80 % of the 1RM ECC for ECC [9], respectively. During the fatiguing exercises, participants performed sets (separated by 25-s of rest) of five unilateral ISO or CONC or ECC contractions (i.e., at an angular velocity of 10°.s⁻¹), separated by 10-s of rest. Whatever the contraction type performed, each contraction duration was ~5s. At the end of each set, a MVIC was performed to evaluate the decline of KF motor performance. The sets of contractions was repeated until a 20 % drop in MVIC compared to PRE was reached on EL. Submaximal isometric contractions were performed at a position combining 40° of hip flexion and 30° of knee flexion (0° = full extension of the hip and the knee). Submaximal concentric contractions started from this position combining 40° of hip flexion and 30° of knee flexion to an ending position combining 65° of hip flexion and 90° of knee flexion. Starting and ending positions were reversed for submaximal eccentric contractions and participants were passively lifted into the starting position by experimenters. During the fatiguing exercises, participants were asked to relax the NEL. The level of electro-myographic activity of the NEL muscles was monitored during the fatiguing exercises of the EL.

## 2.4. Measures

### 2.4.1. Torque production of the knee flexors

In order to evaluate the unilateral force production of the KF in both lower limbs, we utilized a recently developed ergometer (Hamtech device, Human Kinematic, France) [16]. Participants were positioned in a prone lying position and secured to the ergometer (i.e., two elastic bands were placed approximately 5 cm above the sacroiliac joint and below the gluteal fold). Unilateral isometric force production of the KF was measured in a standardized position for both EL and NEL, with the hip flexed at 40° and the knee flexed at 30°. Force transducers inserted into the frame of the ergometer (S-beam, LS02-s, Tech Co. Ltd, Shenzhen, China; capacity: 1 kN) allowed for force production measurement of each lower limb. Each force transducer connected to a lever arm was adjusted vertically according to the participants' anatomical characteristics. A distance of 5 cm above the external malleolus on the Achilles tendon was used to place the contact point of the lever arm. The force signal was recorded using a Biopac MP 150 (Biopac Systems, Inc., Goleta, CA, USA) at a sampling frequency of 1000 Hz. During offline data processing, the force measurements were converted into torque by considering the lever arms of the participants, which refers to the distance between the lateral tibial condyle and the force transducer. Additionally, the ergometer was equipped with a potentiometer (P4500, Novotechnik U.S., Inc., Southborough, MA, USA) that allowed measurement of angular velocity. During dynamic muscle contractions, participants received direct feedback on the angular speed through a screen placed in front of them.

### 2.5. Surface electromyography (sEMG) of the knee flexors

Ag-AgCl pairs (Contrôle-Graphique, Brie-Comte-Robert, France) of electrodes (diameter of 10 mm and an inter-electrode distance of 20 mm) were used to collect sEMG activity of the biceps femoris (BF) muscles in both EL and NEL [17]. A reference electrode was positioned on the lateral tibial condyle on each lower limb. To achieve low-resistance impedance (<3 kΩ), the participants' skin was shaved, and a gentle abrasion was applied using emery paper before placing the electrodes. Indelible ink marks were applied to the skin to ensure consistent and precise repositioning of the electrodes throughout the entire experiment. sEMG signals were sampled at a frequency of 2 kHz, amplified with a bandwidth frequency of 10–500 Hz, a common mode rejection ratio of 110 dB, a Z input of 1000
MΩ, and a gain of 1000. The Biopac MP150 system (Biopac Systems, Inc. in Goleta, CA, USA) was used to record the sEMG signals.

## 2.6. Myostimulation of the knee flexors

Two self-adhesive rectangular electrodes (5 cm × 9 cm - Stimex, Wetzlar, Germany) were placed on both EL and NEL. The cathode was positioned on the hamstrings' proximal part (i.e., below the gluteal fold) and the anode was located at the popliteal fossa. Electrodes positions were marked on the skin to guarantee similar placement across sessions. Percutaneous electrical stimulations of 1-ms duration and 400V maximal output voltage were delivered through those electrodes via an electrical stimulator (type DS7 Digi-timer, Hertfordshire, UK). Maximal amplitude of the BF compound muscle action potential (BF $M_{max}$) and maximal twitch force were determined at rest with progressive increments of the stimulation intensity. To ensure maximal spatial recruitment of motor units during the myostimulation of the KF at PRE, POST and POST24, the stimulation intensity was set at 120 % of the stimulation intensity eliciting single maximal twitch force and BF $M_{max}$ [18].

## 2.7. Measures of perceived motor fatigue and perceived muscle soreness

Perceived motor fatigue has been defined as “a feeling of diminishing capacity to cope with physical stressors” [19]. During the current experiment, participants had to estimate their perceived motor fatigue (PMF) on the French translated and validated version of the Rating-of-Fatigue Scale [20], from 0 (no fatigue) to 10 (total fatigue and exhaustion – nothing left).

Perceived muscle soreness (PMS) was assessed by applying a steady 25-N pressure with a 0.5 cm diameter cylindrical object underneath and above BF sEMG electrodes on both the EL and NEL [21,22]. Participants had to grade their muscle soreness using a visual analog scale from 0 (no pain) to 10 (worst pain). The two obtained values (i.e., taken underneath and above the electrodes) were averaged for analysis.

## 2.8. Data analysis

For both EL and NEL, for all submaximal fatiguing exercises (ISO, CONC, ECC) at any time measurements (PRE, POST, POST24), the greatest MVIC peak torque was kept for data analysis. The sEMG activities of the BF muscles in the EL and NEL were quantified by root mean square (RMS) values calculated over a 500-ms period around MVIC peak torque. BF RMS values obtained were normalized to the respective BF $M_{max}$ recorded at each time measurements (i.e., BF RMS/$M_{max}$). Potentiated paired twitch torque electrically evoked at 100 Hz ($Pt_{100Hz}$) associated with the greatest MVIC was retained for analysis. With the potentiated paired twitch torque electrically evoked at 10 Hz, the corresponding $Pt_{10Hz}$-to-$Pt_{100Hz}$ ratio was computed to examine the possible existence of low frequency fatigue [14,23,24]. These values were considered as peripheral fatigue measures. Central fatigue measures comprised the BF RMS/$M_{max}$ values and the maximal voluntary activation level (VA) of the KF quantified according to the following formula [24,25]:

$$VA (\%) = \left( 1 - \left( \frac{\text{Superimposed } Pt_{100Hz}}{Pt_{100Hz}} \right) \right) \times 100$$

The number of contractions achieved during the unilateral ISO, CONC and ECC fatiguing exercises were collected and the total torque-time integral (TTI) was computed from the area under the torque-time curve. The TTI produced during each set of the fatiguing exercises was calculated for each contraction type (excluding the MVICs realized at the end of each set). Considering the number of sets performed by the participants, each individual total duration of the exercises was divided in three periods accounting for 1/3 of the total duration of the submaximal fatiguing exercises performed. The MVICs performed between sets were linearly interpolated to the nearest values at 33 % and 66 % of the individual total duration. These MVICs were expressed as a relative percentage from the MVIC value produced at PRE. Likewise, RMS values of the BF muscles of the EL and NEL quantified over the entire duration of each contraction of the submaximal exercises and linearly interpolated to the nearest values (i.e., 33 % and 66 % of the individual total duration) were expressed as a relative percentage of the initial value measured at PRE. The MVIC and BF RMS values kept for analysis at the end (i.e., 100 %) of the sets achieved corresponded to the values assessed after the last set of each participant.

## 2.9. Statistical analyses

Analyses were realized using version 26 of IBM SPSS Statistics software for Windows (IBM Corp., Armonk, NY, USA). The Kolmogorov-Smirnov test was used to assess the normality of the distribution of each variable. The comparison of the unilateral fatiguing exercises (ISO, CONC and ECC) was carried out using generalized linear mixed-effect model (GLMM) analyses with linear distribution when the normality of the distribution was confirmed. When the normality of the distribution was not observed, the data were adjusted to the best GLMM fitting model. Models with and without inclusion of a random variable (i.e., participant) were tested using the Akaike information criterion (AIC) and the model showing the smallest score was kept for the analysis [26]. The GLMM analyses performed for MVIC, VA, BF RMS/$M_{max}$, BF $M_{max}$, $Pt_{100Hz}$, $Pt_{10Hz}$-to-$Pt_{100Hz}$ ratio, and PMS scores included the following fixed effects: limb (EL and NEL), muscle contraction type (ISO, CONC and ECC) and time (PRE, POST, POST24). The analysis of PMF scores comprised muscle contraction type and time fixed effects. During the unilateral fatiguing exercises, TTI and %MVIC values were analyzed with fixed effects of muscle contraction type and percentage of exercise duration (33 %, 66 % and 100 %) whereas BF RMS were studied with fixed effects of limb, muscle contraction type and percentage of exercise duration. The number of repetitions performed during the
exercises was tested with a fixed effect of muscle contraction type only. When the model displayed significant interaction or main effects (p < 0.05), Sidak adjusted pairwise comparisons were used. To determine the meaningfulness of significant results, Cohen's d effect sizes (ES) were computed (i.e., 0.2 < small < 0.5; 0.5 < medium < 0.8; large ≥ 0.8). When significant outcomes were observed, Pearson correlation analyses were done to assess the relation between the immediate relative MVIC reduction (POST-PRE) and the relative changes in central and peripheral fatigue measures (i.e., VA, BF RMS/Mmax, BF Mmax, Pt100Hz, Pt10Hz-to-Pt100Hz ratio) as well as with PMF and PMS scores relative changes. The r values of the correlation coefficient obtained were interpreted according to a recent proposal [27]. Unless specified, in the manuscript, in the tables and figures, data are expressed as mean ± standard deviation.

Intraclass correlation coefficients (ICC3,1) with 95 % confidence intervals [28] were computed to indicate the relative reliability of MVIC, Pt100Hz, VA and Pt10Hz-to-Pt100Hz ratio for both EL and NEL measured during the eccentric familiarization session (i.e., Intra-Session) and between values measured before (PRE) the unilateral submaximal isometric, concentric and eccentric exercises (i.e., Inter-Sessions). ICCs >0.90 and between 0.75 and 0.90 indicate excellent reliability and good reliability, respectively. ICCs between 0.5 and 0.75 show moderate reliability, whereas ICCs <0.5 reflect poor reliability [26]. Absolute reliability was calculated as the standard error of measurement (SEM) with SEM values lesser than 10 % of the average measure indicating an excellent absolute variability [29,30]. Finally, to evaluate the data variability for both Intra- and Inter-Sessions, a coefficient of variation (CV) was calculated for the above-mentioned variables. The acceptability threshold of CV was set to 10 % [30,31].

# 3. Results

## 3.1. Unilateral fatiguing exercises

The 1RM CONC and the 1RM ECC measured for all participants was 214.4 ± 55.7 N m and 177.2 ± 41.6 N m, respectively. A significant muscle contraction type effect was found for the number of repetitions performed between fatiguing exercises ($F_{(2,36)} = 8.46; p = 0.001$) with greater repetitions during ECC (40.4 ± 12.7) compared to ISO (28.1 ± 16.4; $p < 0.05$) and CONC (25.8 ± 8.4; $p < 0.01$). However, no significant muscle contraction type effect was observed for the total TTI between the fatiguing exercises ($F_{(2,36)} = 2.56; p = 0.092$). The total TTI produced was $55.9 \pm 28, 46.8 \pm 18.8$ and $59.2 \pm 21$ kN m s for ISO, CONC and ECC, respectively.

During the exercises, a significant percentage of exercise duration effect was observed for %MVIC values ($F_{(3,144)} = 133.18; p < 0.001$). In comparison to baseline, %MVIC values were significantly reduced at 33 % ($-13.94 \pm 7.66\%$; $p < 0.001$), 66 % ($-18.85 \pm 8.36\%$; $p < 0.001$) and at the end ($-27.31 \pm 7.31\%$; $p < 0.001$) of the fatiguing exercises duration (Table 1). In addition, %MVIC values at 66 % were significantly lower than 33 % ($p = 0.001$), and %MVIC values at the end of the exercise duration were significantly smaller than those produced at 33 % and 66 % ($p < 0.001$; Table 1). A significant limb × muscle contraction type interaction ($F_{(2,216)} = 20.95; p < 0.001$) was noted for the BF RMS values during the fatiguing exercises. Regardless of muscle contraction type and percentage of exercise duration, BF RMS values of the EL were greater ($57.74 \pm 16.26\%$; $p < 0.001$; ES = 1.73; Table 1) than values of the NEL ($10.81 \pm 10.26\%$). In addition, irrespective of time, BF RMS values produced during ECC in the EL were lower than ISO and CONC values ($p < 0.001$). Irrespective of the percentage of exercise duration, in the NEL, BF RMS values produced during CONC were higher than the ones produced during ISO ($p = 0.001$) and ECC ($p = 0.02$).

<caption>**Table 1**</caption>

Maximal voluntary isometric contraction (MVIC), torque-time integral (TTI) and electromyographic activity of the biceps femoris (BF RMS) measured during the unilateral submaximal isometric, concentric and eccentric exercises.

<table><thead><tr><th rowspan="2"></th><th rowspan="2">Percentage of exercise duration</th><th rowspan="2">MVIC (%)</th><th rowspan="2">TTI (kN.m.s)</th><th colspan="2">BF RMS (%)</th></tr><tr><th>EL</th><th>NEL</th></tr></thead><tbody><tr><td rowspan="3">ISOMETRIC</td><td>33 %</td><td>86.7 ± 6.3***</td><td>19.32 ± 9.32</td><td><strong>67.1 ± 19.1</strong></td><td>6.2 ± 5.7<sup>£</sup></td></tr><tr><td>66 %</td><td>80.1 ± 7.9***,###</td><td>18.46 ± 9.36</td><td><strong>66.2 ± 18.4</strong></td><td>7.9 ± 6.8<sup>£</sup></td></tr><tr><td>100 %</td><td>72.4 ± 8.0***,###,$$$</td><td>17.84 ± 9.49</td><td><strong>62.6 ± 13.1</strong></td><td>8.3 ± 7.7<sup>£</sup></td></tr><tr><td rowspan="3">CONCENTRIC</td><td>33 %</td><td>83.8 ± 4.2***</td><td>16.44 ± 6.36</td><td><strong>62.5 ± 13.7</strong></td><td>14.6 ± 14.8</td></tr><tr><td>66 %</td><td>78.6 ± 5.5***,###</td><td>15.16 ± 6.14</td><td><strong>61.1 ± 14.7</strong></td><td>16.0 ± 15.8</td></tr><tr><td>100 %</td><td>72.1 ± 6.6***,###,$$$</td><td>15.33 ± 6.86</td><td><strong>61.1 ± 15.2</strong></td><td>14.9 ± 15.4</td></tr><tr><td rowspan="3">ECCENTRIC</td><td>33 %</td><td>87.6 ± 11.0***</td><td>20.49 ± 7.41</td><td><strong>46.3 ± 11.5<sup>a</sup></strong></td><td>9.6 ± 4.8<sup>£</sup></td></tr><tr><td>66 %</td><td>84.7 ± 10.4***,###</td><td>17.96 ± 6.33</td><td><strong>46.7 ± 10.4<sup>a</sup></strong></td><td>10.0 ± 4.7<sup>£</sup></td></tr><tr><td>100 %</td><td>73.6 ± 7.8***,###,$$$</td><td>20.72 ± 8.37</td><td><strong>46.0 ± 11.6<sup>a</sup></strong></td><td>9.9 ± 5.0<sup>£</sup></td></tr></tbody></table>

Irrespective of muscle contraction type, MVIC values (expressed as a percentage of the initial value for each muscle contraction type) measured at 33 %, 66 % and at the end (100 %) of the fatiguing exercises duration were significantly lower from the pre-exercise MVIC (***p < 0.001); measured at 66 % and 100 % were significantly lower from 33 % (###p < 0.001); and measured at 100 % were significantly lower from 66 % ($$$p < 0.001$). In bold ($p < 0.001$), BF RMS values (expressed as a percentage of the maximal BF RMS value of the respective limb) were significantly greater in the exercised limb (EL) compared to the non-exercised limb (NEL).

££ and £ Irrespective of the percentage of exercise duration, BF RMS values measured during ISO ($p < 0.01$) and ECC ($p < 0.05$) in the NEL were lower than CONC values.

a Irrespective of time, BF RMS values measured during ECC in the EL were lower than ISO and CONC values ($p < 0.001$).
## 3.2. MVIC at PRE, POST and POST24

A significant limb × time interaction was observed for MVIC ($F_{(2,216)}$ = 16.17; p < 0.001). MVIC measured at PRE in the EL and NEL did not differ between contraction types (p > 0.05). Regardless of the muscle contraction type, a significant reduction in MVIC was reported between PRE and POST in the EL (−27.73 ± 6.75%; p < 0.001; ES = 1.23; Fig. 2) and in the NEL (−8.53 ± 11.05%; p = 0.003; ES = 0.47; Fig. 2). For both limbs, MVIC measured at POST24 was not different from PRE values (p = 0.09) whatever the muscle contraction type. No significant correlation was noted between MVIC reduction observed in the EL and MVIC reduction evidenced in the NEL (p > 0.05). For reliability of MVIC measures, see Supplementary Material (Table 3).

## 3.3. Peripheral fatigue parameters assessed at PRE, POST and POST24

A significant main time effect was observed for the $Pt_{100Hz}$ ($F_{(2,216)}$ = 5.08; p = 0.007). $Pt_{100Hz}$ measured at PRE in both the EL and NEL did not differ between contraction types (p > 0.05). Regardless of the limb or the muscle contraction type, $Pt_{100Hz}$ significantly decreased between PRE and POST (−7.46 ± 17.20%; ES = 0.37; p = 0.005; Fig. 3). At POST, a significant weak correlation was observed in the EL between $Pt_{100Hz}$ decreases and MVIC losses ($r$ = 0.39, p = 0.014), whereas no significant correlation was found between $Pt_{100Hz}$ decreases and MVIC loss in the NEL (p > 0.05). At POST24, $Pt_{100Hz}$ had returned to PRE values. No limb × muscle contraction type × time interaction was reported on $Pt_{10Hz}$-to-$Pt_{100Hz}$ ratio ($F_{(4,216)}$ = 0.18; p = 0.95; Table 2). No limb × muscle contraction type × time interaction was observed for the BF $M_{max}$ values ($F_{(4,216)}$ = 0.02; p = 1.00; Table 2). For reliability of $Pt_{100Hz}$ and $Pt_{10Hz}$-to-$Pt_{100Hz}$ ratio measures, see Supplementary Material (Table 3).

## 3.4. Central fatigue parameters assessed at PRE, POST and POST24

A significant limb × time interaction was observed for VA ($F_{(2,216)}$ = 14.05; p < 0.001). No significant difference in VA was noted at PRE between contraction types (p > 0.05) in both EL and NEL. In the EL, irrespective of the muscle contraction type, a significant decrease in VA was noted between PRE and POST (−2.60 ± 3.92%; ES = 0.85; p < 0.001; Fig. 4), while VA had returned to PRE values at POST24. No significant modification of VA values was noticed in the NEL. No limb × muscle contraction type × time interaction was observed for the BF RMS/$M_{max}$ values ($F_{(4,216)}$ = 0.53; p = 0.72; Table 2). For reliability of VA measures, see Supplementary Material.

## 3.5. Perceived fatigue assessed at PRE, POST and POST24

A significant time effect was found for the PMF scores ($F_{(2,108)}$ = 86.37; p < 0.001) with increased PMF scores at POST compared to PRE (p < 0.001; ES = 1.77; Fig. 5) regardless of the muscle contraction type. No significant correlation was observed between increased PMF scores and MVIC reduction in the EL or NEL (p > 0.05).

<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Relative maximal voluntary isometric contraction (MVIC; mean ± standard error) decrease in the exercised limb (EL; red bars) and non-exercised limb (NEL; blue bars) after the unilateral submaximal isometric (ISO), concentric (CONC) and eccentric (ECC) exercises. POST-PRE: difference between immediately after and before the exercises. POST24-PRE: difference between 24 h after and before the exercises. *** and ** Irrespective of muscle contraction type, significant MVIC decrease in the EL (p < 0.001) and NEL (p < 0.01) respectively.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Fig. 3.** Potentiated paired twitch torques electrically evoked at 100Hz (Pt<sub>100Hz</sub>) of the EL (exercised limb) and the NEL (non-exercised limb) measured before (PRE; yellow bars), immediately after (POST; orange bars) and 24 h after (POST24; brown bars) the unilateral submaximal isometric (ISO), concentric (CONC) and eccentric (ECC) exercises. Black lines represent individual data. **Irrespective of muscle contraction type and limb, Pt<sub>100Hz</sub> was lower at POST compared to PRE (p < 0.01).**</figcaption></figure>

### 3.6. Perceived muscle soreness assessed at PRE, POST and POST24

Although no limb × muscle contraction type × time interaction was noted for PMS scores, significant main effects of limb ($F_{(1,216)}$ = 6.73; p = 0.01), muscle contraction type ($F_{(2,216)}$ = 4.66; p = 0.01) and time ($F_{(2,216)}$ = 7.60; p = 0.001) were observed. PMS scores reported in the EL were significantly greater than the NEL (p = 0.01; ES = 0.27; Fig. 6). In addition, PMS scores reported after ECC were significantly higher than ISO exercises (p = 0.024; ES = 0.31) and CONC (p = 0.024; ES = 0.31). Finally, irrespective of the limb or the muscle contraction type considered, PMS scores at POST (p = 0.004; ES = 0.57; Fig. 6) and at POST 24 (p = 0.001; ES = 0.56; Fig. 6) were significantly greater than PRE values. At POST, the increase in PMS scores of the EL was correlated with Pt<sub>100Hz</sub> alterations (weak correlation, r = 0.34, p = 0.033) and tended to be correlated with the MVIC loss (weak correlation, r = 0.31, p = 0.058).

## 4. Discussion

This is the first study examining the effects of unilateral ISO, CONC and ECC exercises on the occurrence of crossover fatigue and its underlying mechanisms. The results showed that: i) a targeted motor performance fatigue of the EL led to a similar TTI produced during the ISO, CONC and ECC unilateral exercises and, ii) muscle contraction type (ISO, CONC or ECC) performed during the unilateral fatiguing exercises in the EL did not affect any of the measured variables. Along with the ~28 % MVIC decrease observed in the EL immediately after the exercises, a significant MVIC decline of ~8.5 % was reported in the NEL at POST evidencing crossover fatigue regardless of muscle contraction type (ISO, CONC and ECC). An overall impaired contractile function was evidenced immediately after all unilateral fatiguing exercises in the EL and the NEL. In contrast, central alteration (i.e., voluntary activation) was only observed in the EL irrespective of muscle contraction type. Finally, an increase in perceived motor fatigue was reported immediately after all unilateral fatiguing exercises (ISO, CONC and ECC). Greater perceived muscle soreness was also reported immediately and 24 h after the unilateral fatiguing exercises.

In this study, the unilateral fatiguing exercises of the EL were stopped when the MVIC was decreased by 20 % for each participant, regardless of the muscle contraction type mode (ISO, CONC and ECC). To reach this goal, a greater number of contractions was achieved in ECC in comparison with ISO and CONC but the total amount of TTI produced during the unilateral fatiguing exercises was not statistically different between muscle contraction types. In the present investigation, the amount of immediate MVIC decrease set in the EL knee flexors was in agreement with a recent study performed on knee extensor muscles [14]. In line with their impaired MVIC reported after isometric, concentric and eccentric exercises (i.e., -17.0 %, -21.7 % and -26.2 %, respectively), we observed MVIC decrements of -27.6 ± 15.0 %, -28.2 ± 7.0 % and -27.4 ± 6.5 % after our ISO, CONC and ECC exercises, respectively. These authors also reported that muscle contraction type (i.e., ISO, CONC and ECC) did not influence the motor performance fatigue of the exercised knee extensor muscles after fatiguing exercises matched for the same TTI. However, to date, no study had compared the influence of ISO, CONC and ECC fatiguing exercises on crossover fatigue of the contralateral homologous muscle. Here, a significant MVIC reduction in the NEL was observed at POST only (i.e., overall decrease of 8.5 %; medium ES of 0.47) irrespective of muscle contraction type (i.e., -9.2 ± 8.8 %, -6.1 ± 11.1 % and -10.3 ± 11.2 % after ISO, CONC and ECC exercises, respectively). Consequently, we evidenced a crossover fatigue phenomenon immediately post-exercise after all unilateral exercises. The crossover fatigue magnitudes observed in the current investigation agree with previous studies that have implemented: i) unilateral isometric exercises of the first dorsal interosseous muscle [11] or the knee extensors [3,32] ii) concentric exercises of the knee extensors [33,34] and, iii) eccentric exercises of the knee extensors [8] or the plantar flexors [9]. However, to date, the two studies that have compared the influence of muscle contraction type (i.e., CONC versus ECC) on motor performance fatigue of the non-exercised limb did not evidence crossover fatigue, even though significant MVIC decreases ranging from -13 % to -55 % were observed in the exercised limbs [6,7]. Consequently, the present investigation performed on the KF demonstrated for the first time that crossover fatigue occurred irrespective of
<caption>**Table 2**</caption>

Knee flexors' neuromuscular function of the EL (exercised limb) and the NEL (non-exercised limb) measured before (PRE), immediately after (POST) and 24 h after (POST24) the unilateral submaximal isometric, concentric and eccentric exercises.

<table><thead><tr><th rowspan="2"></th><th rowspan="2"></th><th colspan="3">ISOMETRIC</th><th colspan="3">CONCENTRIC</th><th colspan="3">ECCENTRIC</th></tr><tr><th>PRE</th><th>POST</th><th>POST24</th><th>PRE</th><th>POST</th><th>POST24</th><th>PRE</th><th>POST</th><th>POST24</th></tr></thead><tbody><tr><td rowspan="2">BF RMS/M<sub>max</sub> (a.u.)</td><td>EL</td><td>0.14 ± 0.07</td><td>0.12 ± 0.06</td><td>0.15 ± 0.08</td><td>0.17 ± 0.12</td><td>0.13 ± 0.11</td><td>0.16 ± 0.11</td><td>0.24 ± 0.18</td><td>0.30 ± 0.25</td><td>0.29 ± 0.23</td></tr><tr><td>NEL</td><td>0.02 ± 0.04</td><td>0.02 ± 0.02</td><td>0.02 ± 0.04</td><td>0.01 ± 0.01</td><td>0.02 ± 0.02</td><td>0.02 ± 0.01</td><td>0.10 ± 0.07</td><td>0.09 ± 0.04</td><td>0.08 ± 0.03</td></tr><tr><td rowspan="2">BF M<sub>max</sub>(mV)</td><td>EL</td><td>3.54 ± 1.81</td><td>3.35 ± 1.82</td><td>3.86 ± 2.22</td><td>3.82 ± 2.43</td><td>3.66 ± 2.42</td><td>3.60 ± 2.00</td><td>4.63 ± 3.04</td><td>4.34 ± 3.16</td><td>4.92 ± 2.89</td></tr><tr><td>NEL</td><td>2.97 ± 1.93</td><td>2.86 ± 1.19</td><td>2.76 ± 1.37</td><td>4.91 ± 3.65</td><td>4.61 ± 3.50</td><td>4.06 ± 3.48</td><td>4.51 ± 2.56</td><td>3.99 ± 2.12</td><td>4.29 ± 2.57</td></tr><tr><td rowspan="2">Pt<sub>10Hz</sub>-to-Pt<sub>100Hz</sub> ratio</td><td>EL</td><td>0.74 ± 0.09</td><td>0.69 ± 0.11</td><td>0.69 ± 0.10</td><td>0.73 ± 0.10</td><td>0.69 ± 0.10</td><td>0.72 ± 0.12</td><td>0.72 ± 0.17</td><td>0.73 ± 0.15</td><td>0.76 ± 0.10</td></tr><tr><td>NEL</td><td>0.70 ± 0.12</td><td>0.69 ± 0.10</td><td>0.66 ± 0.08</td><td>0.71 ± 0.08</td><td>0.68 ± 0.12</td><td>0.67 ± 0.09</td><td>0.71 ± 0.11</td><td>0.76 ± 0.20</td><td>0.71 ± 0.09</td></tr></tbody></table>

Root Mean Square values of Biceps Femoris muscle normalized to the respective BF compound muscle action potential (BF RMS/M<sub>max</sub>); Biceps Femoris compound muscle action potential BF M<sub>max</sub>; Pt<sub>100Hz</sub> to Pt<sub>10Hz</sub> ratio.
<figure><img src="image_5.png" /><figcaption>**Fig. 4.** Voluntary activation (VA) of the EL (exercised limb) and the NEL (non-exercised limb) measured before (PRE; yellow bars), immediately after (POST; orange bars) and 24 h after (POST24; brown bars) the unilateral submaximal isometric (ISO), concentric (CONC) and eccentric (ECC) exercises. Black lines represent individual data. *** Irrespective of muscle contraction type, VA at POST was lower than PRE in the EL (p < 0.001).</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>**Fig. 5.** Perceived motor fatigue (PMF) assessed before (PRE; yellow bars), immediately after (POST; orange bars) and 24 h after (POST24; brown bars) the unilateral submaximal isometric (ISO), concentric (CONC) and eccentric (ECC) exercises. Data are presented as median and interquartile range. *** Irrespective of muscle contraction type, PMF scores at POST were greater than PRE (p < 0.001).</figcaption></figure>

the muscle contraction type (ISO, CONC or ECC), although the muscle group studied and/or the fatiguing exercise realized could account for the discrepancy observed with these previous studies [6,7].

In the present investigation, the MVIC decreases reported in both EL and NEL might be partly ascribable to peripheral fatigue, as
<figure><img src="image_7.png" /><figcaption>Fig. 6. Perceived muscle soreness (PMS) in the EL (exercised limb) and NEL (non-exercised limb) measured before (PRE; yellow bars), immediately after (POST; orange bars) and 24 h after (POST24; brown bars) the unilateral submaximal isometric (ISO), concentric (CONC) and eccentric (ECC) exercises. Data are presented as median and interquartile range.</figcaption></figure>

** Irrespective of muscle contraction type and limb, PMS scores were greater at POST and POST24 compared to PRE (p < 0.01).

§ Irrespective of muscle contraction type and time, PMS scores after ECC exercises were greater than ISO and CONC exercises.

££ Irrespective of muscle contraction type and time, PMS scores in the EL were greater than in the NEL (p < 0.01).

evidenced by the immediate (i.e., at POST) impairment of Pt₁₀₀Hz (i.e., -7.5 %; small ES < 0.4). In the EL, the Pt₁₀₀Hz evoked at POST dropped by -9.5 %. These results differ with the 41-46 % immediate alterations of Pt₁₀₀Hz reported in the knee flexors after isometric exercises [35,36]. The fatiguing exercise performed leading to greater maximal voluntary contraction impairment ranging from 27 to 51 % and/or the position adopted by the participants in these studies (i.e., seated on a chair) could partly explained the difference with the present study where unilateral exercises induced ~28 % MVIC decreases and were performed in a prone lying position. However, in these previous studies, the influence of muscle contraction type on knee flexors' peripheral fatigue was not investigated. To date, the influence of muscle contraction type (ISO, CONC and ECC) on peripheral fatigue was only assessed recently in the knee extensors [14]. These authors reported a greater decrement of the potentiated doublet twitch and the Pt₁₀₀Hz-to-Pt₁₀₀Hz ratio immediately after the fatiguing eccentric exercise in comparison with the isometric one [14]. Here, although potentiated doublet twitches were impaired immediately after the fatiguing exercises, no significant difference was noted between muscle contraction types. In addition, we did not observe Pt₁₀₀Hz-to-Pt₁₀₀Hz ratio alterations. The muscle group studied could account for these discrepant observations as evidenced by the lower decrease of single twitch and Pt₁₀₀Hz-to-Pt₁₀₀Hz ratio reported in the knee flexors than in the knee extensors [36]. Considering the small ES reported for Pt₁₀₀Hz loss and the weak correlation (r = 0.39) observed between the immediate MVIC reduction and Pt₁₀₀Hz impairment, the immediate knee flexors motor performance fatigue reported in the EL was partly due to an impaired muscle contractile function.

In the NEL, an immediate reduction (i.e., at POST) of Pt₁₀₀Hz (i.e., -5.4 %) that did not significantly differ from those of the EL was evidenced. In addition, we noted that muscle contraction type (ISO, CONC and ECC) did not influence the peripheral fatigue in the NEL. To our knowledge, an impaired electrically evoked twitch in a contralateral non-exercised homologous muscle was only observed after isometric exercises [3,11]. While considered marginal (i.e., ~2 %) in the knee extensors [3], single twitch impairment of ~20 % was observed in the first dorsal interosseous muscle [11]. In this latter study, the involuntary augmentation of electromyographic activity (i.e., up to 38 % of the maximal activity) and force production (i.e., up to 28 % of the maximal voluntary contraction) in the non-exercised contralateral muscle noted during the ipsilateral muscle fatiguing exercise may have contributed to the impaired electrically evoked twitch. However, in the present study, and although only the biceps femoris muscle was recorded, the BF RMS values of the NEL measured during all unilateral fatiguing exercises (ISO, CONC and ECC) did not exceed ~11 % of its maximal activity. Although we cannot exclude a possible influence of such small involuntary electromyographic activity in our Pt₁₀₀Hz impairments in the NEL, it is highly unlikely that it fully contributed to the ~7.5 % decreases. Indeed, during the fatiguing exercises, the level of electromyographic activity of the EL reached 58 % of its maximal activity and only a ~10 % decrease of Pt₁₀₀Hz was observed. It is extremely challenging to explain Pt₁₀₀Hz alterations observed in our NEL. Previous studies have, for example, suggested that the cardiovascular system could spread the metabolic by-products accumulated in the EL to the NEL [37,38]. In addition, a recent study reported elevated baseline muscle concentration in inorganic phosphate in a resting limb subsequent to a contralateral fatiguing exercise [39]. However, none of these studies provided sound scientific evidence to demonstrate the exact physiological implication of these proposed mechanisms in the reduced performance of the contralateral limb. Another possible explanation for the decreased Pt₁₀₀Hz in the NEL could be related to unpotentiated testing conditions at POST in comparison with PRE. Indeed, PRE measurements
were performed after warming up the NEL, whereas measures at POST were realized after a period of rest due to the duration of the unilateral exercise on the EL. It is known that electrically evoked twitches are potentiated after a conditioning exercise (e.g., like our warm up) and return to control values within approximately 10 min [40,41]. In addition, it has been reported that twitch potentiation is not influenced by the contraction type [42]. Then, considering that the average duration of the exercises performed on the EL was ~9.5 min, it is likely that the NEL was tested in unpotentiated conditions. Future studies should ascertain the influence of unilateral exercise on the electrically evoked twitches of the contralateral non-exercised homologous muscle and to systematically monitor the possible influence of involuntary contraction of the NEL during the ipsilateral fatiguing exercise.

In contrast to peripheral fatigue occurring in both EL and NEL, central fatigue was observed in the EL only, as highlighted by the immediate significant impairment of VA. Moreover, the muscle contraction type (ISO, CONC and ECC) did not influence the decrease of VA (−2.6%; ES = 0.85; large effect). This result is in line with a previous study comparing muscle contraction types on the knee extensors' motor performance fatigue [14]. Although no prior study compared the influence of muscle contraction type, knee flexors' voluntary activation was significantly reduced by 8.4% [35] or remained unchanged [36] after isometric exercises. In the present study, central fatigue was also examined through the measure of BF RMS/Mmax values and no significant modification was found. Consequently, and although the BF muscle does not represent the entire KF muscles' involved in knee flexion, the central alterations observed in the EL may have occurred at both spinal and supraspinal levels since the measurement of VA does not allow for the distinction between these two levels [43]. Indeed, sensory neurons of groups III-IV afferents projecting from the musculoskeletal system to the central nervous system can modulate the descending central drive at both spinal and supraspinal levels to limit peripheral fatigue [44]. Their inhibitory actions originating from an altered muscle homeostasis (i.e., exercise-induced increase of metabolites), evidenced by the Pt100Hz alterations, may have contributed to the development of central fatigue observed in the EL. However, no correlation between MVIC reduction and VA decrease evidenced at POST in the EL was observed in the present investigation. In the NEL, no significant modification of VA was observed. This result contradicts with voluntary activation impairments ranging from ~3.5% to ~15% reported in the non-exercised contralateral homologous knee extensors after isometric fatiguing exercises [3,32] and in the plantar flexors after an eccentric fatiguing exercise [9]. Both fatiguing exercises performed and/or muscle groups studied may account for these conflicting results with our investigation. Therefore, within the limits of the central fatigue parameters examined in the present investigation (i.e., voluntary activation and BF RMS/Mmax values), the motor performance fatigue in the NEL did not seem driven by central fatigue.

In the present investigation, significant greater PMS scores were observed immediately after the exercises (medium ES of 0.57), irrespective of the muscle contraction type and the limb. Our observation contradicts with a study performed on the knee extensors showing that a maximal unilateral eccentric exercise induced greater muscle soreness on the exercised muscle than isometric and concentric exercises matched for the same TTI [14]. Although the muscle group studied could account for these conflicting observations, it has been highlighted that muscle pain can reduce maximal force production as well as voluntary activation [45] of the painful muscle immediately after a fatiguing exercise. Moreover, Norbury et al. [45] emphasized that muscle pain was strongly correlated to both force and voluntary activation alterations supporting that the decrease in performance and central fatigue were mediated by perceived pain. Consequently, an increased perceived pain within a muscle contributes to the force production impairment of the tested muscle through adjustments within the central nervous system. Here, although PMS scores were enhanced in both EL and NEL at POST, we failed to evidence correlations between VA impairments and augmented PMS scores of each limb, but VA was altered in the EL. This observation contradicts with a previous study [45] but the induction and the measurement of pain in this study (i.e., hypertonic saline injection) differed from the present investigation (i.e., exercise-induced muscle soreness). However, we observed that increased PMS scores at POST: i) tended to be correlated to MVIC decrease in the EL (i.e., weak correlation, r = 0.31), and ii) were significantly correlated with Pt100Hz impairment in the EL (i.e., weak correlation, r = 0.34). Therefore, we cannot fully exclude that the impaired MVIC in the EL could not be ascribable to a possible influence of pain-related response. In another interesting experimental approach, evoking pain through hypertonic saline injection in the knee extensors altered the voluntary activation of the contralateral muscles throughout and immediately after a fatiguing exercise [46]. In a recent crossover fatigue study, immediate and prolonged impaired voluntary activation was also reported in the non-exercised contralateral plantar flexors along with an increased muscle soreness evidenced in both exercised and non-exercised limbs [9]. In contrast, and although augmented PMS scores were observed in the present investigation, we failed to demonstrate significant VA alteration in the NEL immediately after all the unilateral exercises. This difference could be partly due to the level of muscle pain reported (i.e., 4.6 ± 2.2) in Norbury et al.'s study [46] in comparison with the low PMS scores rated in the present investigation (i.e., 0.5 ± 0.8), and/or to the different experimental designs (i.e., hypertonic saline injection versus exercise-induced muscle soreness). Consequently, and although the exact mechanism cannot clearly be evidenced in the present investigation, the impaired MVIC in the EL could be ascribed to pain-related response. In contrast, in the NEL, it seems that different factors (i.e., pain-related response and possibly peripheral fatigue) may account for the -8.5% crossover fatigue evidenced.

Another interesting result of the present investigation is the significant increase in PMF scores (large ES of 1.77) observed immediately after all the fatiguing exercises. To the best of our knowledge, no previous study has compared the influence of muscle contraction type on perceived motor fatigue. Although PMF scores do not permit to distinguish perceived motor fatigue between the EL and the NEL, the unilateral fatiguing exercises have induced a global feeling of fatigue. Previous authors have suggested that perceived motor fatigue and motor performance fatigue are interdependent [1,19,47] but, in the present investigation, no correlation was observed between PMF scores and MVIC decreases in both EL and NEL. Similar observations (i.e., absence of correlation between perceived motor fatigue and motor performance fatigue) were recently reported [48]. In this elegant study, performing an exercise with upper limb muscles increased rates of perceived fatigue and decreased the subsequent performance of lower limb muscles. In addition, perceptions of fatigue were correlated with elevated effort perception and diminished affects during the lower limbs
performance. The authors have concluded that perceived fatigue was involved in the perceptual and affective processes responsible for the reduced performance [48]. Therefore, and although effort perception and affective valence were not measured in the present investigation, it can be assumed that the increased global feeling of fatigue reported here could also participate to the crossover fatigue phenomenon. Consequently, like pain perception, exercise-induced perceived motor fatigue may provide valuable information when investigating crossover fatigue and future studies should assess it.

In few crossover fatigue studies, it has been shown that motor performance fatigue of the contralateral homologous muscles could be observed 24 h after a concentric exercise [34] and up to 48 h after eccentric exercises [8,9]. In the present investigation, measurements were performed 24 h after the fatiguing exercises to assess the possible occurrence of prolonged crossover fatigue. In contrast to these studies reporting prolonged crossover fatigue, motor performance fatigue in the present investigation was no longer observed in the EL and NEL at POST24. In addition, PMF scores had returned to PRE values at POST24. At POST24, only a significant increase in PMS scores for both EL and NEL was noted. Although this observation agrees with recent observations involving eccentric exercise [8,9], motor performance fatigue was still present in both the exercised and non-exercised limbs of these studies, which was not the case in the present investigation. Considering that the immediate amount of crossover fatigue reported in these two studies [8, 9] was comparable to the present investigation, we assumed that the muscle group examined accounted for the difference observed in the occurrence of prolonged crossover fatigue.

Some limitations of the present investigation should be pointed out. First, it is important to consider that the present outcomes may be influenced by the muscle group assessed. In fact, a recent study evidenced that knee flexors exhibited different motor performance fatigue magnitude and etiology compared to knee extensors [36]. Second, only sEMG activity of the BF muscle was measured and neural adaptations within other KF muscles (i.e., semitendinosus or semimembranosus) could have occurred. In this context, a study evidenced that high activation imbalance between hamstring muscle heads influenced the time to task failure of a submaximal isometric exercise [49]. In addition, a recent study using shear wave elastography measures demonstrated that the biceps femoris long head was more fatigued than both the semitendinosus and the semimembranosus muscles during eccentric contractions whereas opposite results were reported during concentric contractions [50]. Third, our experimental design involved familiarization sessions composed of numerous isometric and eccentric contractions possibly participating to the low levels of muscle soreness reported here [51]. Finally, the variability of the individual responses to the different fatiguing exercises may participate to the uncertainty of the underlying mechanisms explaining the crossover fatigue in the NEL.

## 5. Conclusion

For a similar relative decrease in force production in the EL following unilateral submaximal KF exercises, an immediate crossover fatigue was observed irrespective of muscle contraction type (i.e., ISO, CONC and ECC). In this context, this study provides new evidence that muscle contraction type may not be a confounding factor participating to the controversy reported in crossover fatigue studies due to the variability in study designs [4,5]. The etiology of motor performance fatigue observed in the EL and in the NEL differed between limbs. Immediate motor performance fatigue in the EL arose from peripheral and central fatigue. In contrary, immediate motor performance fatigue in the NEL was not mediated by central fatigue. Interestingly, our findings evidenced increased perceived motor fatigue (i.e., encompassing perceived muscle soreness) immediately after the exercises. Future studies are needed to ascertain some of the underlying mechanisms proposed.

## CRediT authorship contribution statement

**Jennifer Gioda:** Writing – review & editing, Writing – original draft, Investigation, Formal analysis, Conceptualization. **Enzo Piponnier:** Writing – review & editing, Software, Methodology, Investigation, Conceptualization. **Florian Monjo:** Writing – review & editing, Methodology, Investigation, Conceptualization. **Jonathan Bredin:** Writing – review & editing, Software, Methodology, Investigation. **Baptiste Corcelle:** Writing – review & editing, Methodology, Investigation, Formal analysis. **Flavio Da Silva:** Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis, Conceptualization. **Serge S. Colson:** Writing – review & editing, Writing – original draft, Supervision, Investigation, Funding acquisition, Data curation, Conceptualization.

## Ethics statement

Experimental procedures were conducted in accordance with the Declaration of Helsinki latest version (2013). The investigation was approved by an Ethics Committee (Protection Committee of People for Biomedical Research Southeast III, France; Authorization Number 2020-A02811-38).

## Data availability statement

Data will be made available on request. For requesting data, please write to the corresponding author.

## Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jennifer Gioda reports financial support was provided by French National Research Agency. If there are other authors, they
declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. This study was also supported by the French government, managed by the National Agency of Research as part of the UCAJEDI Future Investments project, reference number ANR-15-IDEX-01.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.heliyon.2025.e42801.

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