Contraction intensity modulates spinal excitability during transcranial magnetic stimulation-evoked silent period in rectus femoris muscle

This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. Author(s): Title: Year: Version: Copyright: Rights: Rights url: Please cite the original version: CC BY 4.0 https://creativecommons.org/licenses/by/4.0/ Contraction intensity modulates spinal excitability during transcranial magnetic stimulation-evoked silent period in rectus femoris muscle © The Author(s) 2023 Published version Gomez-Guerrero, Gonzalo; Ansdell, Paul; Howatson, Glyn; Avela, Janne; Walker, Simon Gomez-Guerrero, G., Ansdell, P., Howatson, G., Avela, J., & Walker, S. (2023). Contraction intensity modulates spinal excitability during transcranial magnetic stimulation-evoked silent period in rectus femoris muscle. European Journal of Applied Physiology, Early online. https://doi.org/10.1007/s00421-023-05367-1 2023 Vol.:(0123456789) 1 3 European Journal of Applied Physiology https://doi.org/10.1007/s00421-023-05367-1 ORIGINAL ARTICLE Contraction intensity modulates spinal excitability duringtranscranial magnetic stimulation‑evoked silent period inrectus femoris muscle GonzaloGomez‑Guerrero1 · PaulAnsdell2 · GlynHowatson2,3 · JanneAvela1 · SimonWalker1 Received: 5 June 2023 / Accepted: 8 November 2023 © The Author(s) 2023 Abstract Purpose Reduced spinal excitability during the transcranial magnetic stimulation (TMS) silent period (SP) has recently been shown to last longer than previously thought in the upper limbs, as assessed via spinal electrical stimulation. Further, there is reason to expect that contraction intensity affects the duration of the reduced spinal excitability. Methods This study investigated spinal excitability at different time delays within the TMS-evoked SP in m.rectus femoris. Fifteen participants performed non-fatiguing isometric knee extensions at 25%, 50% and 75% of maximum voluntary contraction (MVC). Lumbar stimulation (LS) induced a lumbar-evoked potential (LEP) of 50% resting M-max. TMS stimulator output induced a SP lasting ~ 200ms. In each contraction, a LEP (unconditioned) was delivered ~ 2–3s prior to TMS, which was followed by a second LEP (conditioned) 60, 90, 120 or 150ms into the silent period. Five contractions were performed at each contraction intensity and for each time delay in random order. Results Compared to the unconditioned LEP, the conditioned LEP amplitude was reduced (−28 ± 34%, p = 0.007) only at 60ms during 25% of MVC. Conditioned LEP amplitudes during 50% and 75% of MVC were reduced at 60ms (−37 ± 47%, p = 0.009 and −37 ± 42%, p = 0.005, respectively) and 150ms (−30% ± 37%, p = 0.0083 and −37 ± 43%, p = 0.005, respectively). LEP amplitude at 90ms during 50% of MVC also reduced (−25 ± 35%, p = 0.013). Conclusion Reduced spinal excitability is extended during 50% and 75% of MVC. In future, paired TMS-LS could be a potential method to understand changes in spinal excitability during SP (at different contraction intensities) when testing various neurophysiological phenomena. Keywords Lumbar stimulation· Spinal inhibition· Lower limbs· Force production· Cortico-spinal tract Abbreviations AHP Afterhyperpolarization ANOVA Analysis of variance BF Bicep femoris CMEP Cervicomedullary-evoked potential EMG Electromyography GTO Golgi tendon organ H-reflex Hoffmann’s reflex kΩ Kiloohm L1 First lumbar vertebra LEP Lumbar-evoked potential LS Lumbar stimulation MEP Motor-evoked potential M-max Maximum compound action potential Ms Milliseconds MVC Maximal voluntary contraction RC Renshaw cells RF Rectus femoris RI Recurrent inhibition s Seconds SOL Soleus muscle SORE Stimulation offset to return of electromyography SP Silent period TMEP Thoracic motor-evoked potential TMS Transcranial magnetic stimulation Communicated by Toshio Moritani. * Gonzalo Gomez-Guerrero [email protected] 1 NeuroMuscular Research Center (NMRC), Faculty ofSport andHealth Sciences, University ofJyväskylä, Viveca (VIV221), 40700Jyväskylä, Finland 2 Faculty ofHealth andLife Science, Northumbria University, NewcastleUponTyne, UK 3 Water Research Group, North West University, Potchefstroom, SouthAfrica European Journal of Applied Physiology 1 3 Introduction Transcranial Magnetic Stimulation (TMS) applied over the contralateral motor cortex of the muscle targeted, in relaxed and active conditions, produces a muscle action potential that can be recorded by electromyography (EMG) and a muscle twitch. The muscle action potential is referred to as the motor-evoked potential (MEP) and provides information about cortico-spinal excitability (Barker etal. 1985; Day etal. 1989a). In addition, when TMS is applied during voluntary muscle contraction there is an interruption of the background EMG activity after the MEP ( Mills 1988; Day etal. 1989b). This interruption is known as the TMS-evoked silent period (SP) and its duration provides information about inhibition of the cortico-spinal tract (Inghilleri etal. 1993; Triggs etal. 1993; Taylor etal. 1996). For some time, changes in the length of SP have been considered as an indicator of altered intracortical inhibition (Kidgell etal. 2013; Ruotsalainen etal. 2014; Manca etal. 2016; Latella etal. 2017). However, while reduced MEP amplitude, as an indicator of intracortical inhibition, has indeed been shown during the TMS-evoked SP, studies have consistently shown concomitant decreases in spinal excitability 50–100ms after TMS that evokes a ~ 200ms SP (Fuhr etal. 1991; Inghilleri etal. 1993; McDonnell etal. 2006; McNeil etal. 2009). Reduced spinal excitability is possibly due to motor-neuron afterhyperpolarization (AHP) and/or recurrent inhibition (RI) via Renshaw cells (RC), as well as Ia interneuron unloading through reciprocal inhibition (Mills 1988; Fuhr etal. 1991; Ziemann etal. 1993). Interestingly, a recent study showed reduced spinal excitability up to 150ms in the upper limbs after TMS, which was argued to be attributed to an increase in Golgi tendon organ (GTO) activity and muscle spindle unloading (Yacyshyn etal. 2016). Thus, emerging evidence suggests that spinal excitability is modulated over a longer proportion of SP than previously thought. One experimental consideration is that traditional H-reflex methodology used in previous studies (Fuhr etal. 1991; Ziemann etal. 1993) limits the assessment of modified spinal excitability < 100ms, as the measure reflects modified pre-synaptic inhibition. In contrast, direct percutaneous activation of the spinal cord predominantly activates monosynaptic cortico-spinal tract axons (Taylor 2006; McNeil etal. 2013) and can be applied during both submaximal and maximal contractions (Petersen etal. 2002; Škarabot etal. 2019a). It would, therefore, be appropriate to test whether there is reduced spinal excitability at time delays greater than 100ms (Yacyshyn etal. 2016) in the lower-limbs, since previous studies have relied on H-reflex methodology (Ziemann etal. 1993). While spinal responses can be elicited at cervical (cervicomedullaryevoked potential (CMEP)) and thoracic (thoracic motorevoked potential (TMEP)) (Martin etal. 2008) segments of the spine, recent studies suggested that lumbar stimulation (lumbar-evoked potentials (LEP)) are a valid (Škarabot etal. 2019a) and more tolerable (Brownstein etal. 2020) method to study spinal excitability of the lower-limbs. One final consideration is that contraction intensity could affect the duration of the reduced spinal excitability during the TMS-evoked SP. Increases in voluntary torque production increase the tension of the tendon and, consequently, increase GTO activity (Houk etal. 1970). In addition, muscle relaxation rate following TMS is greater with increased torque, which could activate muscle spindles as the sarcomeres lengthen (Vernillo etal. 2022). As such, afferent feedback mechanisms may be modified by increased torque level and potentially influence spinal excitability during SP. In the knee extensors, contractions of 25% of maximal voluntary contraction (MVC) resulted in the unconditioned TMEP being the same amplitude as the subsequent (TMS-) conditioned TMEP evoked at a time delay of 100ms (Finn etal. 2018). In another study, the conditioned TMEP amplitude at a time delay of 100ms was decreased when contracting to 50% of MVC (Brownstein etal. 2020). These results suggest contrasting responses between 25 and 50% of MVC. Examining the contributing factors to the SP in locomotor muscles is important for determining exercise-induced alterations in nervous system function throughout the spectrum of health, exercise and disease (Sidhu etal. 2013). Consequently, there is a need to directly examine the duration of spinal inhibition within the TMS-evoked SP in the lowerlimbs across different contraction intensities. The purpose of the study was to assess spinal excitability at different time delays (60, 90, 120 and 150ms) within the TMS-evoked SP in the rectus femoris (RF) muscle with lumbar stimulation (LS) at different contraction intensities (25, 50, and 75% of MVC). It was hypothesized that reduced spinal excitability would be observed at longer time delays within the SP at increasing contraction intensities. Material andmethods Participants Twenty-two healthy adults (8 female) volunteered for the study. Seven participants were not considered due to possible activation of ventral roots (see Lumbar-evoked potentials). Therefore, the data presented here are representative of the 15 (4 female) volunteers fulfilling all study requirements (males: 11 subjects, 31 ± 6years, height 178 ± 6cm, weight 82 ± 8kg; females: 4 subjects, 28 ± 1years, height 166 ± 8cm, weight 64 ± 7kg). All included participants European Journal of Applied Physiology 1 3 were free from neurological illness and musculoskeletal injury in the lower-limbs for the last 6months, were not taking any medications known to affect the nervous system and had no contraindications to transcranial magnetic stimulation (TMS), which was assessed via a health questionnaire (modified from Rossi etal. (2009). Before testing, all participants were fully informed of the procedures and possible risks, and each participant provided written inform consent. The study was approved by the Ethical committee of the University of Jyväskylä (10.01.2020) and was conducted with accordance with the Declaration of Helsinki (2013). An a priori sample size estimation was conducted using G*Power software (version 3.1, University of Dusseldorf, Germany), based on data presented by Yacyshyn etal. (2016) for α = 0.05 and power = 0.80. The estimated sample size needed was 18 participants to assess torque × time delay interaction between unconditioned and conditioned LEPs. Experimental set‑up Detailed description of Torque, M-max, TMS, Lumbar stimulation and EMG can be found in the subsections below. Participants visited the laboratory on one occasion. To assess responses in the RF muscle, participants were sat in a custom-built chair with a calibrated load cell (Faculty of Sport and Health Sciences, University of Jyväskylä, Finland) with hip and knee at 90° flexion and the shin strapped with a non-elastic restraint ~ 2cm superior to the ankle malleoli. The voltage signal originating from the load cell was calibrated and converted into torque (N·m). All measures were performed on the right (i.e., dominant) leg, assessed by selfreport of which foot they primarily kick a ball (van Melick etal. 2017). Once the participant was secured to the dynamometer, the maximum compound action potential (M-max) was assessed in a relaxed condition. Two maximal voluntary contraction (MVC) trials were performed 60s apart. Prior to the MVC, two contractions at ~ 50% and ~ 80% of estimated MVC were performed as a warm-up. Verbal encouragement and visual feedback were provided to motivate participants to produce maximal effort. Thereafter, target contraction intensities (25%, 50% and 75% of MVC) were displayed on the screen as visual feedback for the participant. Placement of the lumbar stimulation electrodes was assessed to avoid activating spinal nerve roots (see Lumbar-evoked potentials). Thereafter, stimulator intensity was adjusted to produce a LEP of 50% of the M-max at rest, and this stimulation intensity was used throughout the experiment. TMS coil placement was defined as the location producing the largest MEP in the RF, and stimulator output intensity was standardized to evoke ~ 200ms SP from the stimulator artefact to the resumption of the voluntary EMG signal, during brief voluntary contractions at each torque. During the session, unconditioned and conditioned LEPs were delivered during the same voluntary contraction. Unconditioned LEP consisted of a single stimulation delivered at the lumbar level. Conditioned LEPs consisted of a paired stimulation of TMS followed by lumbar stimulation separated by predetermined and randomly ordered time delays (60, 90, 120 and 150ms). Participants were instructed to contract to, and briefly hold, one of the three different contraction intensities (25, 50 and 75% of MVC) in a randomized order. Once the participant reached the required level, an unconditioned LEP was delivered followed by a conditioned LEP at one of the different time delays (Fig.1). The contractions were held for 5–8s and stimuli were delivered 2–3s apart. Sets of five unconditioned, followed by conditioned LEPs, were given per time delay and per torque level as a single block, giving a total of 60 unconditioned and conditioned stimuli. To avoid fatigue (see Results), 30, 45 and 60s rest was given between contractions at 25%, 50% and 75% of MVC, respectively, and 60, 120 and 180s rest was given between the sets of 5 contractions. At the end of the protocol, M-max and MVC were reassessed. Peripheral nerve stimulation Percutaneous electrical stimulation of the femoral nerve (3.2cm cathode/anode arrangement; Polar Neurostimulation Electrodes, Espoo, Finland) was performed to elicit M-max in RF (1ms square pulse duration; Digitimer DS7AH, Hertfordshire, UK). Electrodes were placed 2cm apart and placed at each side of the femoral nerve, located by palpation and identification of the femoral artery (Walker etal. 2016). M-max was elicited by gradually increasing stimulator output intensity until the EMG response plateaued. To ensure supramaximality, this intensity was further increased by 50% (mean ± standard deviation intensity: 257 ± 151mA). Transcranial magnetic stimulation Single TMS pulses were delivered using a Magstim 2002 magnetic stimulator (Magstim Co., Ltd., Whitland, UK) connected to a concave double-cone coil, positioned over the left cortical hemisphere for RF with a posterior-to-anterior current orientation. The hotspot was defined, at rest, as the position eliciting the largest MEP recorded in the EMG using the same intensity (i.e., 50–70% stimulator output) producing a visible MEP. The coil position was marked on the scalp, once the hotspot was found, to maintain the same position throughout the protocol. Stimulus intensities were set to evoke a silent period of ~ 200ms for all contraction intensities (Table1). European Journal of Applied Physiology 1 3 Lumbar‑evoked potentials LEPs were elicited with a constant-current stimulator (1ms square pulse duration; Digitimer DS7AH, Hertfordshire, UK) via self-adhesive electrodes (Polar Neurostimulation Electrodes, Espoo, Finland). The cathode (5 × 10cm) was centered over the first lumbar vertebra (L1) and the anode (circular shape; 3.2cm diameter) was placed on the midline of the vertebral column ~ 5cm above the top edge of the cathode as described by Škarabot etal. (2019a). The intensity of stimulation (309 ± 108mA) was standardized to 50% of the M-max evoked in the resting position. Potential activation of ventral roots was assessed by examining the onset latency of the LEP with an increase in stimulator intensity (Petersen etal. 2002) and tracking LEP amplitude during increased voluntary contraction (Taylor etal. 2002). Should the ventral roots be activated by the stimulation procedures, onset latency would have shortened with an increase in stimulator intensity and LEP amplitude would have been the same during increased voluntary contraction (Petersen etal. 2002; Taylor etal. 2002, 2006; Škarabot etal. 2019a). Dorsal root activation was assessed via paired LS with 50ms time delay (Fig.2), where the amplitude of the second LEP was compared to the first. Evidence of dorsal root activation would be a decrease in the second LEP due to post-activation depression at the motor-neuron pool (Hofstoetter etal. 2018). All remaining participants showed no sign of the responses described and reported that they found LS to be tolerable. Bipolar surface electromyography andtorque Muscle activity was recorded using adhesive Ag/AgCl electrodes (3 × 2cm, BlueSensor N, Ambu, Penang, Malaysia) from m.Bicep Femoris (BF) and RF according to SENIAM Guidelines (Hermens etal. 2000). Skin was shaved, abraded with sandpaper, and wiped with alcohol before setting the electrodes in bipolar arrangement with 2cm center-to-center distance. Impedance was set < 2kΩ, and the reference electrode was positioned above the patella. EMG data were amplified (1000 ×), bandpass filtered (16–1000Hz; Neurolog System, Digitimer Ltd, UK)) and sampled online at 3000Hz using CED Power1401-3 (Cambridge Electronic Design Ltd, Cambridge, UK). Torque was sampled at 1000Hz, amplified by a custombuilt amplifier (ForAmps 1 v1.2, University of Jyväskylä, Fig. 1 One participant’s mean (solid) and individual (dashed) trials that represent the experimental design of one set of unconditioned and conditioned lumbar stimulation at different time delays taken from 25% MVC trials. TMS transcranial magnetic stimulation, LS lumbar stimulation ▸ European Journal of Applied Physiology 1 3 Finland) and converted by a 16-bit A/D board (CED Power1401-3, Cambridge Electronics Design, Cambridge, UK) in combination with Spike2 software (version 6.10, Cambridge Electronic Design, Cambridge, UK). Data andstatistical analyses Offline analyses were performed with Spike software (version 6.10, Cambridge Electronic Design, Cambridge, UK) to manually obtain M-max amplitude, MVC, MEP Silent Period and unconditioned LEP onset latencies. The other outcome measures were analyzed by a customized MATLAB script (version R2020b, The MathWorks, Inc., Natick, USA). Peak-to-peak amplitude of LEPs and MEPs was analyzed automatically between latencies-of-interest following peripheral nerve stimulation, lumbar stimulation or TMS (Taylor etal. 1999), respectively. Torque was averaged over the 100ms before the stimulator artefact. SP duration was determined, through visual inspection, as the time from the stimulator artefact to the return of voluntary EMG (Damron etal. 2008). SPSS software (version 26.0, SPSS Inc., Chicago, USA) was used for all statistical methods. Means and standard deviation (SD) were calculated and reported throughout. Normality of the data was tested with the Shapiro–Wilk test and confirmed by z-score with an acceptance of + 2 to -2 (e.g. skewness score/skewness scoreSE and kurtosis score/ kurtosis scoreSE). Data that did not fulfil those requirements were Log10 transformed, which then fulfilled the requirements for Normality. Paired t-tests were used to assess possible effects of fatigue between M-maxpre and M-maxpost, MVCpre and MVCpost, and to evaluate unconditioned LEP amplitude at different torque levels in the control measurements (shown in Fig.3). One-way analysis of variance (ANOVA) was used to assess potential differences between the three contraction intensities in control measures: Unconditioned LEP latencies, MEP amplitude and MEP Silent Period (shown in Table1). To determine whether Normalized [Conditioned/Unconditioned LEP*100] LEPs responded differently at the tested time delays between the three different torque levels, two-way repeated measures ANOVA was employed. When sphericity assumptions were violated, Greenhouse–Geisser corrections were used. Posthoc Bonferroni adjustments were used when significant main effects were found. When comparing Unconditioned and Conditioned LEP at each time delay, the Benjamin–Hochberg test corrected for multiple paired t test comparisons with a 10% false discovery rate. Effect sizes are represented as partial eta-squared values (ηp2 = small: 0.01, medium: 0.06, large: 0.14) for the factors of the ANOVA and as Hedge’sg for between-group effect sizes for these relative Table 1 Mean and standard deviation values of MEP, lumbar stimulation and involuntary EMG activity parameters from the participants at different submaximal torque levels These values represent the standardization of the measurement MVC maximal voluntary contraction, TMS transcranial magnetic stimulation, MEP motor evoked potential, SP silent period, SORE stimulation offset to return of electromyography, LEP lumbar evoked potential 25% MVC 50% MVC 75% MVC TMS stimulator output (%) 66 ± 16 64 ± 12 65 ± 14 MEP SP: SORE (ms) 216 ± 15 210 ± 10 216 ± 14 MEP (mV) 2.16 ± 1.35 2.02 ± 1.10 1.79 ± 0.84 LEP latency (ms) 6.3 ± 0.7 6.6 ± 0.7 6.6 ± 0.5 Involuntary EMG activity amplitude (mV) 0.11 ± 0.07 0.14 ± 0.09 0.20 ± 0.14 Fig. 2 Data extracted from one participant showing that spinal root activation did not occur. A When increasing the intensity of stimulator output there was no reduction in latency. B A lumbar stimulated doublet with 50ms interval, showing similar amplitudes between the stimulations European Journal of Applied Physiology 1 3 changes (g = small: < 0.3, medium: 0.3–0.8, large: > 0.8). Αlpha was set at 0.05. Results Control measurements There were no statistically significant differences between time delays for MEP amplitude during 25% of MVC (F(3, 56) = 0.033, p = 0.992), during 50% of MVC (F(3, 56) = 0.024, p = 0.995), or during 75% of MVC (F(3, 56) = 0.191, p = 0.902). Additionally, there were no statistical differences between SP duration at any contraction intensity (F(2 42) = 1.110, p = 0.339), indicating standardized conditions throughout the experiment to examine spinal excitability. There were no statistically significant differences between M-maxpre and M-maxpost (M-maxpre = 3.27 ± 1.13mV, M-maxpost = 2.96 ± 1.04mV, p = 0.054, 95% CI [−0.01, 0.62], Hedges’ g = 0.27) nor between MVCpre and MVCpost (MVCpre = 221 ± 60N·m; MVCpost = 214 ± 54N·m, p = 0.106, 95% CI [−1.74, 15.25], Hedges’ g = 0.12). LEP latencies did not show statistical difference between time delays during 25% of MVC (F(3, 56) = 0.106, p = 0.956), during 50% of MVC (F(3, 56) = 0.016, p = 0.997) or during 75% of MVC (F(3, 56) = 0.153, p = 0.902). There was a statistically significant difference between unconditioned LEP amplitude during 25% vs 50% of MVC (p < 0.001, 95% CI [−1.74, 15.25], Hedges’ g = −0.26) and 25% vs 75% (p = 0.001, 95% CI [−0.21, −0.06], Hedges’ g = −0.27) of Fig. 3 Mean (± SD) and individual values of unconditioned LEP response normalized to M-max at different contraction intensities. Increases in LEP amplitude with increases in torque shows that the stimulation was evoked trans-synaptically European Journal of Applied Physiology 1 3 MVC, although no statistical difference was found between 50% of MVC and 75% of MVC (p = 0.956, 95% CI [−0.05, 0.05], Hedges’ g = −0.01) (Fig.3). Collectively, these findings indicate that LS activated the cortico-spinal tract. Effects oftorque onspinal excitability atdifferent time delays Two-way repeated measures ANOVA showed a significant main effect between time delays (F (2,5, 102.4) = 6.542, p = 0.001, ηp2 = 0.135) and torque × time delay interaction (F (4.9, 102.4) = 2.953, p = 0.016, ηp2 = 0.123) for the normalized LEP. Post hoc analyses revealed significant difference in LEP amplitude between 60ms (0.73 ± 0.27) and 150ms (0.95 ± 0.34) (p = 0.007, 95% CI [−0.398, −0.046], Hedges’ g = −0.27) and 90ms (0.75 ± 0.35) and 150ms (p = 0.004, 95% CI [−0.352, −0.050], Hedges’ g = −0.25) during 25% of MVC (Fig.4). Unconditioned vs conditioned LEP Unconditioned LEP was compared to the conditioned LEP at each time delay at the three contraction intensities. During 25% of MVC, conditioned LEP amplitude was statistically lower than unconditioned LEP at 60ms (t(14) = −3.128, p = 0.007, 95% CI [−0.464, −0.087], Hedges’ g = −0.62), but not at 90ms (t(14) = −2.397, p = 0.075, 95% CI [−0.505, −0.028], Hedges’ g = −0.58), 120ms (t(14) = −1.285, p = 0.220, 95% CI [−0.292, 0.073], Hedges’ g = −0.18), nor 150ms (t(14) = 0.722, p = 0.482, 95% CI [−0.248, 0.123], Hedges’ g = −0.13). During 50% of MVC, statistical differences were found at 60, 90 and 150ms (t(14) = −3.052, p = 0.009, 95% CI [−0.634, −0.111], Hedges’ g = −0.76, t(14) = −2.843, p = 0.013, 95% CI [−0.446, −0.062], Hedges’ g = −0.44 and t(14) = −3.099, p = 0.008, 95% CI [−0.502, −0.091], Hedges’ g = −0.52, respectively), where the conditioned LEP was lower than the unconditioned LEP. There were no statistically significant differences in conditioned versus unconditioned LEP amplitude at 120ms (t(14) = −2.073, p = 0.057, 95% CI [−0.451, 0.008], Hedges’ g = −0.36). During 75% of MVC, the conditioned LEP amplitude was significantly lower than unconditioned LEP (Fig.4) at 60ms and 150ms (t(14) = −3.348, p = 0.005, 95% CI [−0.602, −0.132], Hedges’ g = −0.78, and t(14) = −3.377, p = 0.005, 95% CI [−0.610, −0.136], Hedges’ g = −0.70, respectively). But no statistically significant differences were observed at 90ms nor 120ms (t(14) = −2.511, p = 0.067, 95% CI [−0.429, −0.034], Hedges’ g = −0.51 and t(14) = −2.626, p = 0.083 (corrected), 95% CI [−0.394, −0.040], Hedges’ g = −0.52, respectively). Fig. 4 Mean (± SD) and individual values of conditioned LEP normalized to the unconditioned LEP. The dashed line represents the unconditioned LEP amplitude. Any data point or bar below the dashed line represents inhibition and any data or bar above the dashed line represents facilitation of the conditioned LEP. Bars represent the mean values at each contraction intensity and time delay. The circles represent each participant’s data at each contraction intensity and time delay. *p < 0.05 vs unconditioned LEP amplitude European Journal of Applied Physiology 1 3 Discussion This is the first study to directly test spinal excitability at different time delays during TMS-evoked SP, and during different contraction intensities, in the lower-limbs (specifically RF). Our results showed reduced spinal excitability during the first 60ms in RF during all contraction intensities, extending to 90ms at 50% of MVC and further reductions were observed at 150ms during 50 and 75% of MVC. These results conflict with a previous study that used CMEPs during a 25% of MVC contraction in upper limb (Yacyshyn etal. 2016); the conditioned CMEP showed differences from the unconditioned response also at 120 and 150ms after TMS. However, our results agree with early studies conducted using H-reflex methodology in both upperand lower-limbs (Fuhr etal. 1991; Ziemann etal. 1996) despite that H-reflex data could be influenced by changes in presynaptic inhibition, which is absent in our methods. The results suggest that reduced spinal excitability is present but largely limited to ≤ 90ms after TMS in lower-limb muscles, at low contraction intensities (i.e., < 25%of MVC). Nevertheless, differences between upperand lower-limbs have previously been presented by Giesebrecht etal. (2010). They reported a facilitatory response to spinal stimulation in tibialis anterior after 10s MVC, in contrast of spinal inhibition observed by Gandevia etal. (1999) in biceps brachii after 5–10s MVC contraction, discussing different physiological mechanisms in upperand lower-limbs muscles. Compiling the existing literature provides indirect support for the present study’s finding in that contraction intensity influenced the duration of reduced spinal excitability during SP. First, Finn etal. (2018) did not observe reduced spinal excitability at 100ms (TMS induced a 200ms SP), given that the conditioned TMEP was similar to the amplitude of the unconditioned TMEP when standardized to 50% of the M-max (as in the current study). Conversely Brownstein etal. (2021) did observe reduced spinal excitability since both conditioned TMEP and LEP amplitude at 100ms (TMS included 200ms SP) were lower than their respective unconditioned amplitudes, again when spinal stimulation was standardized at 50% of the M-max. As Finn etal. (2018) employed contraction intensities of 25% of MVC, whereas Brownstein etal. (2021) employed 50% of MVC, this suggests that contraction intensity influences the duration of reduced spinal excitability. In directly assessing this hypothesis, spinal excitability was reduced at 60ms but no longer at 90ms after TMS contracting to 25% of MVC, matching the findings of Finn etal. (2018). However, reductions in conditioned LEP were observed at 90ms during 50% of MVC and at 150ms during 50% and 75% of MVC, providing support for and extending the findings of Brownstein etal. (2021). Thus, we suggest that increased contraction intensity modulates spinal excitability distinctly in that reduced stimulation-induced responses are apparent at longer time delays when contracting at a higher intensity. The suggested mechanisms for the decrease in spinal excitability during TMS-evoked SP are: afterhyperpolarization (AHP), recurrent inhibition via Renshaw cells, Ia interneuron unloading through reciprocal inhibition, and/ or GTO inhibition (Mills 1988; Fuhr etal. 1991; Ziemann etal. 1993; Yacyshyn etal. 2016). Although AHP, RI and GTO inhibition are dependent on the preceding motor-neuron activity (Hultborn & Pierrot-Deseilligny 1979; Ziemann etal. 1993) and the size of the conditioned test stimuli (Hultborn & Pierrot-Deseilligny 1979), AHP may not account for more than ~ 56ms, since discharge rate at 50% of MVC is ~ 18 pps in the VL (Kamen & Knight 2004). There is evidence that AHP could impact excitability up to approx. 100ms, depending on motor-neuron firing rate (Piotrkiewicz etal. 2007), as observed in upper-limb muscles. Thus, the exact duration of the influence of AHP is still unresolved in different muscles. However, converging evidence suggests that this may not be the case in explaining the difference between conditioned LEP amplitude during 25% versus 50% of MVC at 90ms in the present study. Among the TMS-evoked SP studies, Ziemann etal. (1993) found that the conditioned/unconditioned H-reflex amplitude progressively decreased with increasing contraction intensity in the soleus muscle (SOL). The authors argued that Renshaw cells might have a stronger influence on TMS-evoked SP inhibition, rather than GTOs or muscle spindles, since the decrease in spinal excitability was ~ 50ms, and those monosynaptic feedback mechanisms start to exert an influence after ~ 40ms in SOL. Although RI may only account for ~ 40ms (Pierrot-Deseilligny & Burke 2005), it could influence discharging rate (Granit etal. 1960). Since stimulator output was not statistically different in 25% and 50% of MVC conditions, a plausible mechanism to explain the prolonged decrease from 60 to 90ms in spinal excitability at higher contraction intensities could be recurrent inhibition via Renshaw cells. In the present study, the interstimulus intervals of 60 and 90ms could also be affected by modified muscle spindle or GTO activity to the cortico-spinal tract. The spindles provide muscle length feedback and GTOs provide tensile feedback (Enoka 2008; Nichols 2018). When there is an increase in contraction intensity, GTOs increase their discharge rate, increasing Ib inhibition (Houk etal. 1970). Further, the TMS-induced muscle twitch has been suggested to also engage GTOs increasing Ib inhibition (Yacyshyn etal. 2016). It is conceivable that the combination of higher intensity contractions and muscle twitch-induced Ib inhibition could be enhanced in the present study’s 50% of MVC trials.