The authors have no acknowledgements.

The experimental procedures outlined received Institutional ethical approval and are in accordance with the Declaration of Helsinki. Data were collected from a representative participant who was aware of the procedures and gave his written informed consent.
1. Instrument Preparation
<ol>
	<li>Clean the skin at the electrode location by shaving, and remove the dirt with alcohol to obtain low impedance (&#60;5 k&#937;).</li>
	<li>Place two AgCl surface electrodes (recording diameter of 10 mm) at 2/3 of the line between the medial condylis of the femur to the medial malleolus for the soleus muscle; on the most prominent bulge of the muscle for the medial gastrocnemius; at 1/3 of the distance along a line between the head of the fibula and the heel for the lateral gastrocnemius; and at 1/3 of the distance along a line between the tip of the fibula and the tip of the medial malleolus for the tibialis anterior muscle, with an interelectrode distance (center-to-center) of 2 cm, according to the SENIAM recommendations30. 
	Note: The soleus muscle electrodes have to be positioned under the distal insertion of gastrocnemii muscles to ensure that they are not recording activity from the heads of the gastrocnemii muscles (cross-talk).</li>
	<li>Place a reference electrode in a central position on the same leg (between stimulation and recording sites).</li>
	<li>Adjust the height and the depth of the chair to obtain an ankle angle of 90&#176; (0&#176; = full plantar flexion), so that the soleus and gastrocnemii muscles are not stretched and the H-reflex is not altered11,12.
	<ol>
		<li>Set the knee angle at 90&#176; (0&#176; = full knee extension) due to the biarticular nature of the gastrocnemii muscles. However, the optimal ankle angle to perform a maximal voluntary torque of the plantar flexors is 70-80&#176; (0&#176; = full plantar flexion)26. Thus, ankle angle will depend on the parameter of interest (electrophysiological versus mechanical recordings). 
		Note: Regardless of the chosen initial angle, it must remain constant throughout the experiment to standardize neuromuscular excitability11,12,27,28.</li>
		<li>Pay particular attention when monitoring the subjects&#8217; posture during the test to maintain constant cortico-vestibular influences on the excitability of the motor pool29.</li>
	</ol>
	</li>
	<li>Firmly strap the ankle to an ergometer, with the anatomical axis of the joint (external malleolus) aligned with the axis of rotation of the ergometer25.
	<ol>
		<li>Have the subject exert pressure on a footplate attached to the ergometer to record plantar flexor torque. Keep the foot immobile throughout the experiment so that small changes in torque can be detected.</li>
	</ol>
	</li>
	<li>Note: Under certain circumstances, the heel may lift slightly off the force plate if the foot and ankle are not secured, which may lead to an incomplete transmission of the torque against the plate. Figure 3 presents a description of the experimental setup.</li>
</ol>
<img alt="Figure 3" src="/files/ftp_upload/52974/52974fig3.jpg" /> 
Figure 3: Experimental setup. Classical experimental setup to record electromyographic (EMG) and torque signals.
<ol start="6">
	<li>Connect the electrodes to the amplifier with cables.</li>
	<li>Set the sampling rate for torque and EMG measurements to 2-5 kHz. Record the EMG signal using an Analog-to-Digital (AD) conversion system. The signal is displayed on a monitor with a data acquisition system, which instantaneously gives values of several parameters (e.g. maximal value, peak-to-peak amplitude, duration). The spectrum of the EMG signal can range between 5 Hz and 2 kHz frequencies, but is mainly contained between 10 Hz and 1 kHz31. Thus, sampling frequency must be high enough to preserve signal shape during EMG recording. Amplify and filter EMG signals (gain = 500-100) using a bandwidth frequency between 10 Hz and 1 kHz8,21,32.</li>
	<li>Place the anode for the electrical stimulation over the patellar tendon.</li>
	<li>Determine the best stimulation site of the posterior tibial nerve to obtain an optimal soleus H reflex for a given intensity, using a hand-held cathode ball electrode in the popliteal fossa. Test several stimulation sites with the cathode ball electrode until a maximal value of the H reflex is reached.
	<ol>
		<li>Record tibialis anterior EMG activity to ensure that the common peroneal nerve is not activated to avoid influence from antagonist Ia afferents12. Set the pulse width at 1 msec to provide an optimal activation of the nervous fibers, especially afferent fibers10.</li>
	</ol>
	</li>
	<li>Place a self-adhesive AgCl cathode at the location of the stimulation site to ensure constant stimulus condition (e.g. pressure, orientation). 
	Note: All of these parameters (subject position, electrode location and stimulation site) do not change for the assessment of the different electrophysiological measurements. Only the intensity of the stimulation and the condition (rest versus contraction) vary.</li>
</ol>
2. Testing Procedures at Rest
<ol start="1">
	<li>Instruct the subject to remain relaxed and to keep his/her muscles at rest.</li>
	<li>Adjust the stimulation intensity to obtain maximal soleus H-reflex amplitude (Hmax; usual range: 20-50 mA). An M-wave of the soleus muscle can be observed at Hmax intensity. 
	Note: For repeated measurements (e.g. before and after a fatiguing protocol), the optimal intensity to obtain a Hmax response may vary during the session. As keeping a constant intensity can lead to an underestimation of Hmax amplitude, it is recommended that the experimenter regularly reevaluates Hmax intensity33.</li>
	<li>Record a minimum of 3 soleus H-reflex responses at this intensity with a minimum interval of 3 sec to avoid post-activation depression34. 
	Note: Although recording several responses is more suitable due to the particular sensitivity of the H-reflex, a single stimulation may be enough under some circumstances, for example when trying to avoid the effects of rapid recovery (e.g. during a fatiguing protocol).</li>
	<li>Increase the stimulation intensity to obtain maximal soleus M-wave amplitude (Mmax; usual range: 40-100 mA). Usually, set the increment in stimulation intensity at 2-4 mA, with an interval of 8-10 sec between two stimuli12,35. The desired intensity is reached when Mmax is obtained, and no H-reflex response can be observed.</li>
	<li>Set the final intensity to 120-150% of Mmax stimulus intensity to ensure that the M-wave attains a plateau of its maximal value. This intensity is called supramaximal intensity in the instructions below.</li>
	<li>Keep constant stimulation intensity for soleus M-wave recordings throughout the session.</li>
	<li>Record 3 soleus M-waves and 3 associated twitch torques at this intensity.</li>
</ol>
3. Testing Procedures During Voluntary Contraction
<ol start="1">
	<li>As a warm-up, ask the subject to perform 10 brief and non-fatiguing submaximal contractions of the plantar flexor muscles, with a few seconds rest between each of the contractions. At the end of the warm-up, take a minimum 1 min rest to avoid any fatiguing effects11.</li>
	<li>Continuously record triceps surae EMG activity. Recording soleus and gastrocnemii muscles allows the analysis of the behavior of different muscle typologies for a single stimulation site24.</li>
	<li>Instruct the subject to perform an isometric maximal voluntary contraction (MVC) of the plantar flexors. The subject has to push as hard as possible against the ergometer by contracting his plantar flexor muscles. Give visual feedback to the subject during the effort, and standardized verbal encouragement19. The MVC is reached when a plateau is observed.</li>
	<li>Deliver a paired stimulation (100 Hz frequency) at supramaximal intensity during the plateau of the MVC (superimposed doublet), and another paired stimulation when the muscle is fully relaxed immediately after the contraction (potentiated doublet) to evaluate the voluntary activation level. Deliver this paired stimulation through a specific device (e.g. Digitimer D185 MultiPulse Stimulator) or through a stimulation program associated with a single pulse stimulator.</li>
	<li>Instruct the subject to perform a second MVC of the plantar flexor with at least 1 min rest between each trial11. If the peak torque from the second trial is not within 5% of the first, additional trials must be performed36. The greatest torque achieved by the subject is taken as the MVC torque.</li>
</ol>
4. Data Analysis
<ol start="1">
	<li>Data Analysis at Rest
	<ol>
		<li>Select a time window including the EMG response associated with the twitch at rest (H-wave or M-wave).</li>
		<li>Measure the peak-to-peak amplitude, peak-to-peak duration, and/or the area of the waves (Figure 4A). If the amplitude is not directly provided by the software, subtract the minimum to the maximum values.
		<ol>
			<li>For the duration, measure the time frame starting from the maximal peak and ending to the minimal peak. For the area, calculate the integral of EMG signal starting from the beginning of the wave and ending to the end of the wave. 
			Note: Peak-to-peak amplitude can reflect: 1) neuromuscular transmission, 2) motor unit action potential amplitude and/or 3) temporal dispersion of motor unit action potential37. M-wave duration reflects neuromuscular propagation37.</li>
		</ol>
		</li>
		<li>For multiple trials, calculate the average of the waves. If the average cannot be directly provided by the software, use spreadsheet software (e.g. the formula function in a spreadsheet program) to calculate this value from several trials (at least 3).</li>
		<li>Select the resting twitch.</li>
		<li>Measure the peak torque associated with the resting twitch (Figure 4B).</li>
		<li>For multiple trials, calculate the average peak torque of the resting twitches. If the average cannot be directly provided by the software, use spreadsheet software (e.g. the formula function in a spreadsheet program) to calculate this value from the several trials (at least 3).</li>
		<li>Repeat these procedures described in point 4.1.2 for the other desired parameters (contraction time or half-relaxation time). The analysis of twitch parameters provides indications as to the excitation-contraction coupling efficiency17. In particular, contraction time provides an index of contraction kinetics8, which can depend upon the chosen muscle group38.</li>
		<li>Calculate the ratio between the peak torque and the sum of M-waves using spreadsheet software (e.g. Excel), to quantify the electromechanical efficiency (Pt/M). As the mechanical responses evoked by posterior tibial nerve stimulation correspond to the activation of the triceps surae as a whole, amplitudes of soleus and gastrocnemii M-waves must be summed39.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 4" src="/files/ftp_upload/52974/52974fig4.jpg" /> 
Figure 4: Explanation of electrophysiological and mechanical responses. (A) Measurement of peak-to-peak amplitude (mV), latency (ms) and area (mV.ms) of a typical M-wave. (B) Measurement of peak twitch torque (Nm), contraction time (ms) and half-relaxation time (msec) of a twitch. <a href="https://www.jove.com/files/ftp_upload/52974/52974fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Data analysis in contraction
	<ol>
		<li>Select a 500 msec time window of soleus EMG activity during the plateau of MVC torque including the peak torque but excluding the time between the stimulation artefact and the end of the silent period of EMG. The silent period corresponds to the suppression of the ongoing voluntary EMG activity following stimulation.</li>
		<li>If the root mean square (RMS) is not directly provided by the software, calculate the RMS to quantify EMG activity using the following formula40: RMSEMG 
		<img alt="Equation 1" src="/files/ftp_upload/52974/52974eq1.jpg" /></li>
		<li>Measure or calculate the RMS of Mmax at rest over the duration of the wave.</li>
		<li>Calculate the RMSEMG/RMSMmax ratio using spreadsheet software. RMSEMG value and RMSMmax value have to be selected from the same muscle.</li>
		<li>Measure the maximal peak torque of the MVC from the baseline of torque at rest to the maximal value of MVC excluding the superimposed torque induced by the doublet stimulation (Figure 5).</li>
		<li>Measure the superimposed torque induced by the doublet stimulation during the MVC, from the voluntary torque value at the onset of the stimulation to the peak of the evoked response (Figure 5).</li>
		<li>Select the potentiated doublet.</li>
		<li>Measure the peak torque associated with the potentiated doublet.</li>
		<li>Calculate the voluntary activation level (VAL) using the following formula40: 
		<img alt="Equation 1" src="/files/ftp_upload/52974/52974eq2.jpg" /></li>
	</ol>
	</li>
</ol>
<img alt="Figure 5" src="/files/ftp_upload/52974/52974fig5.jpg" /> 
Figure 5: Measurement of superimposed and potentiated doublet on mechanical signal. To record the superimposed peak torque (Pts), stimulation doublet is evoked during the plateau of isometric maximal voluntary contraction (MVC). To record potentiated peak torque (PtP), stimulation doublet is evoked at rest after the offset of MVC.

The authors have nothing to disclose.

Percutaneous nerve stimulation enables the quantification of numerous characteristics of the neuromuscular system not only to understand the fundamental control of neuromotor function in healthy humans, but also to be able to analyze acute or chronic adaptations through fatigue or training17. This is very beneficial especially for fatiguing protocols, where measurements must be performed as soon as possible after exercise end to avoid the effects of rapid recovery42.
Alth...

Percutaneous electrical nerve stimulation is widely used to assess neuromuscular function1. The basic principle consists of inducing an electrical stimulus to a peripheral motor nerve to evoke a muscular contraction. Mechanical (torque measurement) and electrophysiological (electromyographic activity) responses are simultaneously recorded. Torque, recorded at the considered joint, is assessed using an ergometer. The electromyographic (EMG) signal recorded using surface electrodes has been demonstrated to represent the activity of the muscle2. This non-invasive method is not painful and more easily implemented than intramuscular recordings. Both monopolar and bipolar electrodes can be used. The monopolar electrode configuration has been shown to be more sensitive to changes in muscle activity3, which can be useful for small muscles. However, bipolar electrodes have been shown to be more effective in improving the signal-to-noise ratio4 and are most commonly used as a method of recording and quantifying motor unit activity. The methodology described below will focus on bipolar recordings. EMG activity is an indicator of the efficacy and integrity of the neuromuscular system. The use of percutaneous nerve stimulation offers further insights into neuromuscular function, i.e. changes at muscular, spinal, or supra-spinal level (Figure 1).
<img alt="Figure 1" src="/files/ftp_upload/52974/52974fig1.jpg" /> 
Figure 1: Overview of the neuromuscular measurements. STIM: nerve stimulation. EMG: Electromyography. VAL: Voluntary Activation Level. RMS: Root Mean Square. Mmax: Maximal M-wave amplitude.
At rest, the compound muscle action potential, also called M-wave, is the short-latency response observed after stimulus artefact, and represents excitable muscle mass by the direct activation of motor axons leading to the muscle (Figure 2, number 3). M-wave amplitude increases with intensity until reaching a plateau of its maximal value. This response, called Mmax, represents the synchronous summation of all motor units and/or muscle fiber action potentials recorded under the surface EMG electrodes5. The evolution of the peak-to-peak amplitude or wave area is used to identify alterations of neuromuscular transmission6. Changes in the mechanical responses associated with the M-wave, i.e. peak twitch torque/force, may be due to alterations in muscle excitability and/or within the muscle fibres7. The association of Mmax amplitude and peak twitch torque amplitude (Pt/M ratio) provides an index of electromechanical efficiency of the muscle8, i.e. mechanical response for a given electrical motor command.
<img alt="Figure 2" src="/files/ftp_upload/52974/52974fig2.jpg" /> 
Figure 2: Motor and reflexive pathways activated by nerve stimulation. Electrical stimulation of a mixed (motor/sensory) nerve (STIM) induces a depolarization of both motor axon and Ia afferent firing. Depolarization of Ia afferents towards the spinal cord activates an alpha motoneuron, which in turn evokes an H-reflex response (pathway 1+2+3). Depending upon the stimulus intensity, motor axon depolarization evokes a direct muscular response: M-wave (pathway 3). At maximal M-wave intensity, an antidromic current is also generated (3&#39;) and collides with reflex volley (2). This collision partially or totally cancels the H-reflex response.
The H-reflex is an electrophysiological response used to assess changes in the Ia-&#945; motoneuron synapse9. This parameter can be assessed at rest or during voluntary contractions. H-reflex represents a variant of the stretch reflex (Figure 2, number 1-3). The H-reflex activates motor units monosynaptically recruited by Ia afferent pathways10,11, and can be subjected to peripheral and central influences12. The method of evoking a H-reflex is known to have a high intra-subject reliability to assess spinal excitability at rest13,14 and during isometric contractions15.
During a voluntary contraction, the magnitude of the voluntary neural drive can be assessed using the amplitude of the EMG signal, generally quantified using the Root Mean Square (RMS). RMSEMG is commonly used a means of quantifying the level of excitation of the motor system during voluntary contraction (Figure 1). Because of the intra- and inter-subject variability16, RMSEMG has to be normalized using the EMG recorded during a muscle-specific maximal voluntary contraction (RMSEMGmax). In addition, because changes in EMG signal may be due to alterations at peripheral level, normalization using a peripheral parameter such as M-wave is required to assess only the central component of EMG signal. This can be done by dividing the RMSEMG by the maximal amplitude or the RMSMmax of the M-wave. Normalization using RMSMmax (i.e. RMSEMG/RMSMmax) is the preferred method as it takes into consideration the possible change of the M-wave duration17.
Motor commands can also be evaluated by calculating the voluntary activation level (VAL). This method uses the twitch interpolation technique18 by superimposing an electrical stimulation at Mmax intensity during a maximal voluntary contraction. The extra-torque induced by stimulating the nerve is compared to a control twitch produced by identical nerve stimulation in a relaxed potentiated muscle19. To evaluate maximal VAL, the original twitch interpolation technique described by Merton18 involves a single stimulus interpolated over a voluntary contraction. Recently, the use of paired stimulation has become more popular because the evoked torque increments are larger, more readily detected, and less variable compared to single stimulation responses20. The VAL provides an index of the capacity of the central nervous system to maximally activate the working muscles21. Currently, VAL evaluated using the twitch interpolation technique is the most valuable method of assessing the level of muscle activation22. Furthermore, peak torque assessed using an ergometer is the most properly studied strength testing parameter applicable of use in research and clinical settings23.
Electrical nerve stimulation can be used in a variety of muscle groups (e.g. elbow flexors, wrist flexors, knee extensors, plantar flexors). However, nerve accessibility makes the technique difficult in some muscles groups. The plantar flexor muscles, especially triceps surae (soleus and gastrocnemii) muscles, are frequently investigated in the literature24. Indeed, these muscles are involved in locomotion, justifying their particular interest. The distance between stimulation site and recording electrodes allows the identification of the different evoked waves of the triceps surae muscles. The superficial part of the posterior tibial nerve in the popliteal fossa and the large number of spindles make it easier to record reflex responses compared to other muscles24. For these reasons, the currently presented reflex methodology focuses on the triceps surae group of muscles (soleus and gastrocnemius). The aim of this protocol is therefore to describe percutaneous nerve stimulation technique to investigate neuromuscular function in the triceps surae.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Biodex dynamometer</td>
			<td>Biodex Medical System Inc., New York, USA</td>
			<td></td>
			<td><a href="http://www.biodex.com/">www.biodex.com</a></td>
		</tr>
		<tr>
			<td>MP150 Data Acquisition System</td>
			<td>Biopac Systems Inc., Goleta, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acknowledge 4.1.0 software</td>
			<td>Biopac Systems Inc., Goleta, USA</td>
			<td></td>
			<td><a href="http://www.biopac.com/">www.biopac.com</a></td>
		</tr>
		<tr>
			<td>DS7A constant current high voltage stimulator</td>
			<td>Digitimer, Hertfordshire, UK</td>
			<td></td>
			<td><a href="http://www.digitimer.com/">www.digitimer.com</a></td>
		</tr>
		<tr>
			<td>Silver chloride surface electrodes</td>
			<td>Control Graphique Medical, Brie-Comte-Robert, France</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Computer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 Cable for connecting the Biodex to the MP150</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 Cable for connecting the Digitimer to the MP150</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 Cable for connecting the MP150 to the computer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of neuromuscular function using percutaneous electrical nerve stimulation

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels). The present protocol describes how this method can be used to stimulate the posterior tibial nerve that activates plantar flexor muscles. Percutaneous electrical nerve stimulation consists of inducing an electrical stimulus to a motor nerve to evoke a muscular response. Direct (M-wave) and/or indirect (H-reflex) electrophysiological responses can be recorded at rest using surface electromyography. Mechanical (twitch torque) responses can be quantified with a force/torque ergometer. M-wave and twitch torque reflect neuromuscular transmission and excitation-contraction coupling, whereas H-reflex provides an index of spinal excitability. EMG activity and mechanical (superimposed twitch) responses can also be recorded during maximal voluntary contractions to evaluate voluntary activation level. Percutaneous nerve stimulation provides an assessment of neuromuscular function in humans, and is highly beneficial especially for studies evaluating neuromuscular plasticity following acute (fatigue) or chronic (training/detraining) exercise.

Increasing stimulus intensity leads to a different evolution of response amplitudes between H- and M-waves. At rest, the H-reflex reaches a maximum value before being totally absent from EMG signal, while M wave progressively increases until reaching a plateau at maximal intensity (see Figure 4 for a graphical depiction of the M-wave and Figure 6 for the evolution of M-waves and H-reflex with intensity). For the soleus muscle, the latency between the stimulus onset and M-wave is about 10...

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Assessment of Neuromuscular Function Using Percutaneous Electrical Nerve Stimulation

We present a protocol to assess changes in neuromuscular function. Percutaneous electrical nerve stimulation is a non-invasive method that evokes muscular responses. Electrophysiological and mechanical properties of these responses permit the evaluation of neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels).

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, ...

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21.6K Views.  Univ. Bourgogne Franche–Comté. We present a protocol to assess changes in neuromuscular function. Percutaneous electrical nerve stimulation is a non-invasive method that evokes muscular responses. Electrophysiological and mechanical properties of these responses permit the evaluation of neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels). 