Eur J Appl Physiol (2011) 111:2451–2459 DOI 10.1007/s00421-011-2047-4 O R I G I N A L A R T I CL E Transcutaneous neuromuscular electrical stimulation: inXuence of electrode positioning and stimulus amplitude settings on muscle response M. Gobbo · P. GaVurini · L. Bissolotti · F. Esposito · C. Orizio Received: 15 February 2011 / Accepted: 8 June 2011 / Published online: 30 June 2011 © Springer-Verlag 2011 Abstract The aim of the study was to investigate the inXuence of two diVerent transcutaneous neuromuscular electrical stimulation procedures on evoked muscle torque and local tissue oxygenation. In the Wrst one (MP mode), the cathode was facing the muscle main motor point and stimulus amplitude was set to the level eliciting the maximal myoelectrical activation according to the amplitude of the evoked electromyogram (EMG); in the second one (RC mode), the electrodes were positioned following common reference charts for electrode placement while stimulus amplitude was set according to subject tolerance. Tibialis Anterior (TA) and Vastus Lateralis (VL) muscles of 10 subjects (28.4 § 8.2 years) were tested in speciWc dynamometers to measure the evoked isometric torque. The EMG and near-infrared spectroscopy probes were placed on muscle belly to detect the electrical activity and local metabolic modiWcations of the stimulated muscle, respectively. The stimulation protocol consisted of a gradually increasing frequency ramp from 2 to 50 Hz in 7.5 s. Compared to RC mode, in MP mode the contractile parameters (peak twitch, tetanic torque, area under the torque build-up) and the metabolic solicitation (oxygen consumption and hyperemia due to metabolites accumulation) resulted signiWcantly higher for both TA and VL muscles. MP mode resulted also to be more comfortable for the subjects. Based on the assumption that proper mechanical and metabolic stimuli are necessary to induce muscle strengthening, our results witness the importance of an optimized, i.e., comfortable and eVective, stimulation to promote the aforementioned muscle adaptive modiWcations. Communicated by R. Bottinelli. Keywords Neuromuscular electrical stimulation · Motor point · Near-infrared spectroscopy · Muscle strengthening This article is published as part of the Special Issue Cluster on the XVIII Congress of the International Society of Electrophysiology and Kinesiology (ISEK 2010) that took place in Aalborg, Denmark on 16–19 June 2010. M. Gobbo (&) · C. Orizio Department of Biomedical Sciences and Biotechnologies, University of Brescia, Brescia, Italy e-mail: gobbo@med.unibs.it M. Gobbo · P. GaVurini · L. Bissolotti · C. Orizio Laboratory of Neuromuscular Rehabilitation (LaRiN), Domus Salutis, Brescia, Italy P. GaVurini Faculty of Motor Sciences, University of Verona, Verona, Italy F. Esposito Department of Sport, Nutrition and Health Sciences, University of Milan, Milan, Italy Introduction Skeletal muscles develop tension to accomplish diVerent functions: besides being motor system actuators, they are responsible for posture maintenance and they contribute as active factors to joint functional stability. Electrical stimulation, by exploiting the adaptive potential of skeletal muscles to increased loads, is widely applied to maintain, improve or restore muscle trophism, and therefore function, in sports practice and rehabilitation settings (Hainaut and Duchateau 1992; Lake 1992; Fluck and Hoppeler 2003; MaYuletti 2006). For the aforementioned purposes, stimulation of impaired or unimpaired muscles predominantly adopts skin-adhesive pad electrodes placed 123 2452 over the target muscles to provide a transcutaneous neuromuscular electrical stimulation (TNMES). TNMES generates an electric Weld between the two surface electrodes and creates a current Xow from anode to cathode into the tissue: for innervated muscles, the current evokes nervous action potentials from the intramuscular unmyelinated motor axon terminal branches and hence elicits muscle Wber contraction (Salmons 1994; Farina et al. 2004); concomitantly, current stimulates sensory nervous structures leading TNMES to be generally coupled to the sensation of discomfort or pain (Reilly 1992) and to be plausibly ill tolerated by subjects. The occurrence of discomfort or pain represents a major limiting factor for the strength development with TNMES (Selkowitz 1985) as the operator/user is forced to lower the stimulus amplitude which determines the amount of simultaneously active MUs and therefore the entity of the evoked tension (Davies et al. 1985). As a consequence, the bearable low stimulation intensities may be insuYcient to induce muscle adaptive responses since, by analogy with voluntary exercise (Salmons 1994), the beneWcial eVects of TNMES are obtained by an adequate intensity of contraction able to provide a proper workload (mechanical stress) and an increased local oxygen consumption (metabolic stress) (Hainaut and Duchateau 1992; Fluck and Hoppeler 2003). Indeed, in their review Fluck and Hoppeler (2003) described the role of the mechanical factor, i.e., the degree of tension, in activating the multiple pathways that inXuence gene expression linked to muscle hypertrophy, as well as the role of several metabolic perturbations occurring in severely exercised skeletal muscle that serve as control in transcriptional induction. Among metabolic stimuli, local tissue hypoxia has been assumed to be an important sensing mechanism that links muscle metabolic perturbations to plastic remodeling of the muscle. For this, both mechanical output and indexes of metabolic stress should be evaluated when assessing the eVectiveness of a particular strengthening modality. Although TNMES has gained a well established role as main tool or in adjunction to other training or therapeutic regimes within rehabilitation and sports practice (Hainaut and Duchateau 1992; Snyder-Mackler et al. 1994; Stevens et al. 2004; Callaghan and Oldham 2004; Talbot et al. 2003; MaYuletti 2006), the procedures adopted by diVerent operators vary considerably incurring the risk of disparate and inconsistent outcomes. As a matter of fact, out of some basic studies on neuromuscular electromechanical features (e.g., KnaXitz et al. 1990; Merletti et al. 1990, Farina et al. 2004, Gobbo et al. 2006, Minetto et al. 2008) in which the electrical stimulation is delivered via the main motor point (MP), TNMES is commonly applied by referring to charts for surface electrode placement or atlases illustrating motor point standard locations which quite markedly diVer one from each other (Botter et al. 2011). Moreover, it is widespread to 123 Eur J Appl Physiol (2011) 111:2451–2459 set the amplitude of stimulation according to subject tolerance, which might be substantially conditioned by several factors: anatomical (inter-individual variability in the spatial distribution of sensory and motor Wbers), physiological (individual level of tolerance to electrical stimuli and adaptation over time) and psychological. In other words, at present, a standard procedure for electrode positioning and amplitude settings is lacking. As a consequence, systematic comparisons between diVerent procedures are essential to identify the most eVective TNMES setting up. Based on the hypothesis (Watson 2000) that the methodological procedures aVect the physical eVects of TNMES (i.e., the distribution of the electric Weld and its relative density through muscle tissue and on motor-sensory nerve structures) which, in turn, inXuence the physiological response, aim of the study was to compare two diVerent procedures: in the Wrst one, the cathode was facing the muscle main motor point and stimulus amplitude was set to the level eliciting the maximal myoelectrical activation according to the entity of the evoked electromyogram (EMG); in the second one, electrodes were positioned following common legally marketed reference charts for electrode placement while stimulus amplitude was set according to subject tolerance. With reference to the previously cited principle stating that mechanical and metabolic stress represent the stimuli needed for strengthening adaptation to occur, the study focuses on acute eVects of the two above-mentioned TNMES procedures, applied on diVerent human leg muscles, in terms of the entity of the evoked muscle mechanical response and the related local oxygen expenditure and regional blood supply. Methods Subjects Ten subjects (six males, four females; age 28.4 § 8.2 years), without neurological or orthopedical diseases, gave their informed consent to participate to the study after being given a full explanation of the experimental procedure according to the Declaration of Helsinki (1964). The subjects had no previous experience with similar experiments and underwent the two designed trials of stimulation (see later for details) 1 week before the actual experimental session in order to be accustomed to the sensation of TNMES. The local Ethical Research Committee approved the proposed experimental design. Measurements For each subject, the dominant Tibialis Anterior (TA) and Vastus Lateralis (VL) were investigated. The leg was Wxed Eur J Appl Physiol (2011) 111:2451–2459 2453 Fig. 1 Experimental setup for TA (left panels) and VL (right panels) muscles. MP mode (top panels) and RC mode (bottom panels) stimulation setups are presented. Black circles indicate cathodes while gray circles indicate anode electrodes. The NIRS probe is marked with N; the EMG probe is marked with E in a speciWcally designed dynamometer equipped with a load–cell (Interface, model SM-500 N, linear response between 0 and 500 N). Load–cell signal was sampled at 1,024 Hz and the isometric torque exerted by the muscle was oZine calculated. To evaluate the electrical activity of the stimulated muscle (M-wave) the electromyogram (EMG) was detected by a probe, placed on the muscle belly, with a linear array of four silver bars electrodes 0.5 cm spaced. The single diVerential EMG from the two middle bars was ampliWed, Wltered (bandpass: 10–512 Hz) and recorded at 1,024 samples/s. A near-infrared spectroscopy (NIRS) system (NIRO-200, Hamamatsu Photonics K.K, Japan) was employed to study local muscle oxygenation and perfusion dynamics: the optode assembly was secured to the skin surface close to the EMG probe and covered with an optically dense sheet to minimize intrusion of ambient light. NIRS measures relative changes in the oxygenation status of hemoglobin (the concomitant minor myoglobin changes can be neglected) and in the total hemoglobin amount that is related to regional blood Xow (Cettolo et al. 2007). The NIRO-200 device refreshed the analog output signal with a 6 Hz frequency. The four NIRS signals were sampled by the same analog-to-digital converter used for torque and EMG signals. Experimental procedures Subjects underwent two experimental sessions, one for TA and one for VL. In each experimental session, bipolar stimulation was administered to the investigated muscle follow- ing two diVerent procedures. In the Wrst one (MP mode), the cathode electrode (5 £ 5 cm) was placed on the main motor point (see Fig. 1, top panels). The motor points (MPs) have been deWned as the superWcial points of stimulation with the lowest motor threshold, i.e., the points on the skin over the muscle belly at which the smallest amount of current is required to produce muscle contraction (Delitto and Robinson 1989; KnaXitz et al. 1990; Lions et al. 2004). Accordingly, the main MP was identiWed, while stimulating at 2 Hz and gradually increasing the stimulation intensity, by moving a pen electrode (area = 1 cm2) on the skin overlying the target muscle until a location providing a minimal detectable mechanical response was found. Subsequently, the amplitude of the stimulus was set to the one providing the maximal M-wave response, i.e., the compound muscle action potential for that MP, and was termed AMPmax. The overall procedure took nearly 2–4 min for each subject. The anode electrode (5 £ 5 cm for TA; 10 £ 5 for VL) was positioned distally over the muscle–tendon junction for TA stimulation and on the proximal fourth of the thigh for VL stimulation (see Fig. 1). In the second procedure (RC mode), a physical therapist was asked to place the cathode electrode (see Fig. 1, bottom panels) according to the most common electrode positioning reference charts available on the market, while stimulation amplitude was set to the maximum level tolerated by the subject. The mentioned most common electrode position areas for TA and VL are shown in Fig. 2 and were inferred from a collection of 12 legally marketed charts (Acuneeds, Australia; Anne Hartley Agency, USA; Beauty 123 2454 Eur J Appl Physiol (2011) 111:2451–2459 that has been used in a previous paper by Orizio et al. (2004) to investigate the mechanical behavior of human TA muscle, allows to study the muscle response from low to more eYcient high stimulation rates thus mimicking the natural increase in the Wring rate of the recruited MUs. This pattern represents a well-tolerated tool to assess the various muscle responses and has also been preferred to the much more common constant frequency tetanic stimulation in light of the possibility to reach high mechanical and metabolic responses in a few seconds, avoiding the rapid onset of fatigue induced by sustained tetanic stimulation rates. After administration of the frequency ramp, a 3 min recovery period was allowed in each trial. Comfort evaluation At the end of each stimulation, subject perception of discomfort was quantiWed by a numeric rating scale (NRS): the subjects were asked to indicate the level of discomfort on a scale ranging from no pain (score = 0) to worst possible pain (score = 10). Fig. 2 Most common cathode locations for TNMES of TA and VL muscles inferred from a collection of 12 legally marketed charts. Circle size is proportional to the number of charts indicating the same location for cathode placement Center Biosan, Biosan World Corporation S.r.l., Italy; Compex®, Compex Medical SA, Switzerland; Cosmogamma®, Cosmogamma S.r.l., Italy; Globus, Globus Italia S.r.l., Italy; IntelliSTIM®, Beac Biomedical, Italy; Neurotrac™, Verity Medical Ltd, UK; CefarCompex®, Djo LLC, USA; Samms®, Italy; Tesmed®, Tesmed, Italy; WinForm®, WinForm S.r.l., Italy) by digitally overlapping the scans from the diVerent sources and matching the anatomical reference points (software for graphical editing: Adobe® Photoshop®, Adobe Systems Incorporated, CA, USA). As suggested by the selected manufacturer’s instructions, the anode electrode (5 £ 5 cm) was placed proximally and close to the cathode for TA while, for VL, the anode (10 £ 5 cm) was kept on the proximal fourth of the thigh (see Fig. 1). Signal analysis From the torque traces recorded during the frequency ramp administration in MP mode and RC mode, the following contractile parameters were investigated: the peak twitch (pT) at 2 Hz, the tetanic tension at 50 Hz (T50), and the tension–time area (TT area) calculated as the area between baseline and the tension signal. To study the metabolic perturbations induced by the contractions elicited with the two stimulation modalities, the proWles of the NIRS parameters, i.e., the oxy-hemoglobin (O2Hb) and deoxy-hemoglobin (HHb) changes, the tissue oxygenation index (TOI), and the normalized total hemoglobin index (THI), were considered. With reference to baseline (average value during a 30 s rest just before stimulation) the values corresponding to the largest negative variation of O2Hb, TOI and THI (THImin) dynamics during contraction and the values corresponding to the largest positive variation of HHb and THI (THImax) during recovery were calculated. Statistics Stimulation protocols The stimulation pattern consisted of a frequency ramp with stimulation rate linearly increasing from 2 to 50 Hz in 7.5 s in order to retrieve information from both single twitch and tetanic torque levels. Stimulus characteristics were: biphasic square wave; 100 s duration. The pattern, 123 Data are reported as mean § standard deviations (SD) for each trial. Paired t test was performed (SigmaPlot, Systat Software Inc.) to identify, for each muscle, signiWcant diVerences in the analyzed parameters between the two investigated trials. SigniWcance level was set to p < 0.05. Eur J Appl Physiol (2011) 111:2451–2459 Torque (N·m) MP mode RC mode 6 4 2 0 O2Hb (µmol/L) - HHb (µmol/L) Fig. 3 Torque traces and related NIRS proWles, from a representative subject, recorded during the frequency ramp contraction and during the recovery phase. The signals refer to TA stimulation in MP mode (left panels) and RC mode (right panels). Dashed lines indicate baselines used as reference for parameter changes quantiWcation 2455 10 5 0 -5 -10 TOI (%) 70 60 50 THI (relative value) 40 1.2 1.1 1.0 0.9 0.8 0 5 10 15 20 0 Time (s) Results 10 15 20 Time (s) Table 1 Mechanical parameters from MP mode and RC mode stimulations of TA and VL muscles Mechanical parameters Typical isometric torque traces obtained from TA during the frequency increasing ramp are reported in Fig. 3. The entity of the mechanical signals resulted lower in RC mode with respect to MP mode. In Table 1, for each muscle, the mean (§SD) pT, T50 and TT area values obtained in MP mode and RC mode trials are reported. All the parameters showed signiWcant diVerence (p < 0.05) between the two stimulation modalities. 5 pT (Nm)* T50 (Nm)* TT area (Nm s)* TA MP 0.68 § 0.43 4.78 § 3.44 59.6 § 42.3 RC 0.48 § 0.28 3.23 § 2.22 38.6 § 25.2 MP 1.68 § 0.87 11.3 § 5.65 131.1 § 72.6 RC 1.15 § 0.89 7.6 § 5.45 72.2 § 54.4 VL Mean (N = 10) and SD values are reported * SigniWcant diVerence (p < 0.05) between modalities NIRS parameters Figure 3 shows typical changes in NIRS parameters during contraction and recovery phase. In Table 2, the averaged (§SD) minimum values for O2Hb, TOI and THI (THImin) and the averaged (§SD) maximum values for HHb and THI (THImax) in response to TA and VL stimulation are presented. All the investigated parameters were signiWcantly diVerent (p < 0.05) in the two applied stimulation 123 2456 Eur J Appl Physiol (2011) 111:2451–2459 Table 2 NIRS parameters changes in MP mode and RC mode stimulations of TA and VL muscles O2Hb (mol/l)* HHb (mol/l)* TOI (%)* THImin (r.v.)* THImax (r.v.)* MP ¡13.9 § 7.6 7.4 § 4.8 ¡11.1 § 6.6 ¡0.19 § 0.12 0.09 § 0.05 RC ¡10.1 § 7.5 4.1 § 3.4 ¡8.5 § 6.7 ¡0.15 § 0.11 0.05 § 0.03 MP ¡10.5 § 4.1 5.9 § 1.8 ¡13.5 § 3.4 ¡0.14 § 0.06 0.04 § 0.03 RC ¡5.8 § 3.3 1.8 § 0.9 ¡5.9 § 2.9 ¡0.07 § 0.02 0.03 § 0.02 TA VL Mean (N = 10) and SD values are reported. THImin (largest negative variation from baseline during contraction) and THImax (largest positive variation from baseline during recovery) are reported in relative values (r.v.) with respect to 1 For each parameter there was a signiWcantly (*p < 0.05) lesser variation from baseline in RC mode with respect to MP mode Table 3 NRS scores for pain evaluation collected after each trial Subjects Tibialis Anterior Vastus Lateralis MP mode RC mode MP mode RC mode 1 0 3 0 1 2 0 2 0 3 3 0 0 0 0 4 0 3 0 3 5 0 6 0 0 6 1 8 0 4 7 0 0 0 6 8 0 2 0 0 9 1 3 1 3 10 0 5 0 2 Mean § SD 0.2 § 0.42 3.3 § 2.54 0.1 § 0.31 2.2 § 1.98 Score 0, no discomfort or pain; score 10, the worst possible pain SigniWcant diVerence (p < 0.05) was present between MP mode and RC mode for both the investigated muscles modalities, indicating lower deviations from baseline in RC mode with respect to MP mode. Discomfort/pain perception NRS scores for comfort level evaluation are presented in Table 3. During MP mode trials, eight subjects indicated no change in their comfort level, whereas two subjects for TA and one subject for VL reported mild discomfort. During RC trial most of the subjects complained of discomfort or frank pain. NRS scores resulted signiWcantly higher (p < 0.05) in RC mode than in MP mode for both the investigated muscles. Discussion In this work, the inXuence of two diVerent muscle transcutaneous electrical stimulation procedures on the evoked 123 muscular activity was studied. The work focused on the analysis of torque and NIRS signals and demonstrated that MP mode evoked larger mechanical and metabolic stress in the muscle. As a consequence, based on Fluck and Hoppeler (2003) review, which described the role of mechanical factors and metabolic perturbations in conditioning skeletal muscle tissue, our results indicate that the stimulation procedure might be a key factor in determining muscle functional improvements induced by TNMES. Unlike RC modality, which is adopted by most professionals and non-professional users, MP mode elicited signiWcantly greater mechanical responses evaluated by the peak tension during single twitches, the maximum tetanic tension and the area under the torque build-up during the frequency ramp. In particular, according to Celichowski et al. (1998) the tension–time area has been proven to be a proper parameter for the estimation of the actual work performed by the contracting motor units and therefore for predicting the eVectiveness of muscle contraction. Evidence exists that this area is proportional to the energy utilized by the contracting skeletal muscle Wbers during brief periods of isometric activity (De Haan et al. 1986; Jobsis and DuYeld 1967; Lochynski et al. 2007). On this basis, an optimal stimulation is obtained when the maximal area during contraction is reached: this was the case of MP modality, indicating a signiWcantly greater workload achieved by the muscle when stimulated on the main MP at AMPmax. Given the analogy of muscle adaptive responses to increased tendon load by either volitional or stimulated muscle actions (Salmons 1994), it is safe to consider that the amount of the evoked muscle contractile work is crucial to determine a proper conditioning eVect for TNMES as well. In fact, Atha (1981) concluded that “muscles strengthen in response to an appropriate stress”, Lai et al. (1988) demonstrated that high training intensities (50% of the maximal voluntary contraction) produced greater strength gains than low training intensities (25% of the maximal voluntary contraction) and MacDougall (1992) stated more speciWcally that muscle mass increases after Eur J Appl Physiol (2011) 111:2451–2459 working at intensities exceeding 60–70% of their forcegenerating capacity. To sum up, the higher the contractile activity, i.e., the mechanical stress, achieved during the stimulation regime, the greater the chance to exceed the threshold levels of activity required to induce muscle strengthening adaptation. The improved mechanical outcomes attained in MP mode trials are explainable by the nearly total absence of discomfort or pain during the stimulation (see later) which is known (Selkowitz 1985) to represent a major limiting factor for TNMES eVectiveness. Poor unpleasant sensory manifestations allow the technique to deliver high current doses which, from the existing linear relationship between stimulus amplitude and tension output (Davies et al. 1985; MaYuletti 2010), suit a maximized activation of the stimulated muscle portion. MP mode resulted also in signiWcantly greater changes in the investigated NIRS parameters with respect to RC mode. From a metabolic standpoint, the energy required to maintain the ATP turnover during muscle contraction is initially derived from immediate intramuscular phosphocreatine stores, which are subsequently restored in the recovery phase by oxidative metabolism (Hamaoka et al. 2007). The higher the oxygen consumption after contraction, the greater the energy expenditure during the previous contractile activity. HHb dynamics studied with NIRS is well accepted to describe local muscle oxygen consumption (Chiappa et al. 2008): in MP mode, the more pronounced HHb increase during recovery suggests a greater metabolic solicitation. Furthermore, considering that the regulation of muscle perfusion depends on an auto-adaptation phenomenon proportional to the local release of metabolites (Dyke et al. 1995), the hyperemic response observed in the recovery phase by studying the THI proWles (Cettolo et al. 2007) indicates a stronger metabolic stress induced by MP mode stimulation. The decrease in TOI (tissue oxygen saturation) during contraction has been attributed to the enhanced cellular oxygen uptake by mitochondria as a consequence of the increased metabolism of the active motor units and to the increased intramuscular pressure that reduces muscle blood supply and oxygen delivery to muscle Wbers (Cettolo et al. 2007; Jensen et al. 1999). In the current study, the signiWcantly larger decrease in TOI coupled with the pronounced decrease in THI obtained in MP mode indicated that the contraction was associated with a prominent drop in muscle O2 tension probably due to either a greater local oxygen consumption and a reduced oxygen supply caused by the increased intramuscular pressure. Since local tissue hypoxia represents an important stimulus for muscle tissue adaptation, a stimulation modality able to induce a more evident hypoxic state in the muscle, like MP mode, should be preferred in the light of the purposes of TNMES to improve muscle trophism and function. 2457 Finally, in parallel with the sought mechanical and metabolic stress increases, the resulting NRS scores indicated a much lower discomfort/pain perception during stimulation in MP mode, thus assuring the maximum level of comfort for the subject. The issue of discomfort/pain due to surface electrical stimulation becomes particularly considerable when sustained electrically evoked contractions need to be induced, typically for rehabilitation purposes, leading some subjects to totally reject TNMES as a treatment option and others not to tolerate the optimal or necessary stimulus amplitude and treatment duration. Selkowitz (1985) advocates that in order to strengthen skeletal muscle using ES the relative increase in strength “may be determined by the ability of the subject to tolerate longer and more forceful contractions”. According to Table 3, most of the subjects referred no discomfort during the MP mode stimulation while the presence of a little discomfort or even frank pain was constant in RC mode. This remarkable diVerence likely relates to the peripheral nerve sensory/motor-speciWc subdivision pattern: the distinct path of the motor nerve branches would then underlie the possibility of current injection via MP (without exceeding AMPmax) to induce a strong selective excitation of the axonal terminations belonging to the motor trunk branched out from the mixed spinal nerve, with a poor or absent concurrent depolarization of somatosensitive nerve Wbers. In favor of this explanation are some recent and preliminary data from our group that resulted from experiments aimed at clarifying which nervous structures are exactly excited during TNMES with diVerent modalities, indicating that AMPmax current injection through areas adjacent to MP constantly involved the simultaneous direct excitation of aVerent somatosensory nerve fascicles, as attested by the peculiar quality of the perceived pain, namely radiating, electric and tingling. It is known that many individuals can develop tolerance to highamplitude painful TNMES after several days of familiarization (Stevenson and Dudley 2001), but it is reasonable to expect that, using an optimized (comfortable and eVective) TNMES procedure as MP mode, subjects might exploit muscle adaptive responses from the early beginning of the treatment program with no need for an initial period of familiarization. In this line, the time required (2–4 min or even less in subsequent sessions) to locate the main MP and to search for AMPmax before the administration of the stimulation would likely result in the enhancement of the outcomes of TNMES. To note that, a proper stimulus amplitude setting, in order to maximally recruit the MUs of the stimulated MP without exceeding AMPmax, represents a keypoint when selective stimulation of a speciWc muscle or muscle group is needed. Indeed, this issue becomes crucial when TNMES is applied to recover the balance between agonist and antagonist muscle functions, a particular condition in which the possible activation and strengthening of 123 2458 the antagonists, resulting from excessively spreading current, should be prevented to avoid counterproductive eVects. Last consideration pertains to the activation of central nervous structures during peripheral TNMES: an increased and dose-dependent activity of speciWc neural areas involved in motor control and somatosensory perception such as primary sensorimotor cortex, cerebellum and cingulate gyrus (Smith et al. 2003) has been recently demonstrated. As reviewed by MaYuletti (2010), this feature might have a relevant role in enhancing restoration or improvement of neuromuscular eYciency and, in line with the current intensity dependence of the mentioned central contributions, MP mode should again be expected to maximize speciWc, in particular cerebellar and motor cortical, supraspinal eVects of TNMES. Conclusions The results from this study conWrm the inXuence of electrode positioning on discomfort/pain perception during TNMES (Lions et al. 2004) and deepen the relation between stimulation sites and the entity of the evoked mechanical and metabolic muscle responses. In particular, stimulation in the more advanced MP mode was found to be considerably more bearable, torque-producing and energy-demanding, leading to greater mechanical and metabolic stress that represent the basic factors for muscle strengthening adaptation. These are relevant Wndings since TNMES of leg muscles has widespread application in research and clinical settings: the evidences from this study might be taken into account to promote the methodological improvements and standardization of the technique allowing subjects to fully tolerate and beneWt from TNMES treatments and leading professionals to attain more consistent and comparable outcomes between trials. References Atha J (1981) Strengthening muscle. Exerc Sports Sci Rev 9:1–73 Botter A, Oprandi G, Lanfranco F, Allasia S, MaYuletti NA, Minetto MA (2011) Atlas of the muscle motor points for the lower limb: implications for electrical stimulation procedures and electrode positioning. Eur J Appl Physiol (accepted) Callaghan MJ, Oldham JA (2004) Electric muscle stimulation of the quadriceps in the treatment of patellofemoral pain. Arch Phys Med Rehabil 85(6):956–962 Celichowski J, Grottel K, Bichler E (1998) The area under the record of contractile tension: estimation of work performed by a contracting motor unit. Acta Neurobiol Exp 58:165–168 Cettolo V, Ferrari M, Biasini V, Quaresima V (2007) Vastus lateralis O2 desaturation in response to fast and short maximal contraction. Med Sci Sports Exerc 39:1949–1959 123 Eur J Appl Physiol (2011) 111:2451–2459 Chiappa GR, Borghi-Silva A, Ferreira LF, Carrascosa C, Oliveira CC, Maia J, Gimenes AC, Queiroga F Jr, Berton D, Ferreira EM, Nery LE, Neder JA (2008) Kinetics of muscle deoxygenation are accelerated at the onset of heavy-intensity exercise in patients with COPD: relationship to central cardiovascular dynamics. J Appl Physiol 104:1341–1350 Davies CTM, Dooley P, McDonagh MJN, White MI (1985) Adaptation of mechanical properties of muscle to high force training in man. J Physiol 365:277–284 De Haan A, De Jong J, Van Doorn EJ, Huijing AP, Woittiez DR, Westra GH (1986) Muscle economy of isometric contractions as a function of stimulation time and relative muscle length. PXugers Arch 407:445–450 Delitto A, Robinson AJ (1989) Electrical stimulation of muscle: techniques and applications. In: Snyder-Mackler L, Robinson AJ (eds) Clinical electrophysiology: electrotherapy and electrophysiologic testing. Williams & Wilkins, Baltimore, pp 95–138 Dyke CK, Proctor DN, Dietz NM, Joyner MJ (1995) Role of nitric oxide in exercise hyperaemia during prolonged rhythmic handgripping in humans. J Physiol 488:259–265 Farina D, Blanchietti A, Pozzo M, Merletti R (2004) M-wave properties during progressive motor unit activation by transcutaneous stimulation. J Appl Physiol 97:545–555 Fluck M, Hoppeler H (2003) Molecular basis of skeletal muscle plasticity–from gene to form and function. Rev Physiol Biochem Pharmacol 146:159–216 Hainaut K, Duchateau J (1992) Neuromuscular electrical stimulation and voluntary exercise. Sports Med 14:100–113 Hamaoka T, McCully KK, Quaresima V, Yamamoto K, Chance B (2007) Near-infrared spectroscopy/imaging for monitoring muscle oxygenation and oxidative metabolism in healthy and diseased humans. J Biomed Opt 12(6):62–105 Jensen BR, Jørgensen K, Hargens AR, Nielsen PK, Nicolaisen T (1999) Physiological response to submaximal isometric contractions of the paravertebral muscles. Spine 24(22):2332–2338 Jobsis FF, DuYeld JC (1967) Force, shortening and work in muscular contraction: relative contributions to overall energy utilization. Science 156:1388–1392 KnaXitz M, Merletti R, De Luca CJ (1990) Inference of motor unit recruitment order in voluntary and electrically elicited contractions. J Appl Physiol 68:1657–1667 Lai HS, De Domenico G, Stauss G (1988) The eVect of diVerent electro-motor stimulation training intensities and strength improvement. Aust J Physiother 34:151–164 Lake DA (1992) Neuromuscular electrical stimulation. An overview and its application in the treatment of sports injuries. Sports Med 13:320–336 Lions GM, Leane GE, Clarke-Moloney M, O’Brien JV, Grace PA (2004) An investigation of the eVect of electrode size and electrode location on comfort during stimulation of the gastrocnemius muscle. Med Eng Phys 26:873–878 Lochynski D, Celichowski J, Korman P, Raglewska P (2007) Changes of motor unit contractile output during repeated activity. Acta Neurobiol Exp 67:23–33 MacDougall JD (1992) Hypertrophy or hyperplasia. In: Komi PV (ed) Strength and power in sport. Blackwell Science, Oxford, pp 230–238 MaYuletti NA (2006) The use of electrostimulation exercise in competitive sport. Int J Sports Physiol Perform 1(4):406–407 MaYuletti NA (2010) Physiological and methodological considerations for the use of neuromuscular electric stimulation. Eur J Appl Physiol 110:223–234 Merletti R, KnaXitz M, De Luca CJ (1990) Myoelectric manifestations of fatigue in voluntary and electrically elicited contractions. J Appl Physiol 69:1810–1820 Eur J Appl Physiol (2011) 111:2451–2459 Minetto MA, Botter A, Ravenni R, Merletti R, De Grandis D (2008) Reliability of a novel neurostimulation method to study involuntary muscle phenomena. Muscle Nerve 37(1):90–100 Orizio C, Gobbo M, Diemont B (2004) Changes of the force-frequency relationship in human tibialis anterior at fatigue. J Electromyogr Kinesiol 14(5):523–530 Reilly JP (1992) Sensory responses to electrical stimulation. In: Electrical stimulation and electropathology. Cambridge University Press, New York, pp 231–284 Salmons S (1994) Exercise, stimulation and type transformation of skeletal muscle. Int J Sports Med 15(3):136–141 Selkowitz DM (1985) Improvement in isometric strength of the quadriceps femoris muscle after training with electrical stimulation. Phys Ther 65(2):186–196 Smith GV, Alon G, Roys SR, Gullapalli RP (2003) Functional MRI determination of a dose–response relationship to lower extremity neuromuscular electrical stimulation in healthy subjects. Exp Brain Res 150(1):33–39 2459 Snyder-Mackler L, Delitto A, Bailey SL, Stralka SW (1994) Use of electrical stimulation to enhance recovery of quadriceps femoral muscle force production in patients following anterior cruciate ligament reconstruction. Phys Ther 74:901–907 Stevens JE, Mizner RL, Snyder-Mackler L (2004) Neuromuscular electrical stimulation for quadriceps muscle strengthening after bilateral total knee arthroplasty: a case series. J Orthop Sports Phys Ther 34(1):21–29 Stevenson SW, Dudley GA (2001) Dietary creatine supplementation and muscular adaptation to resistive overload. Med Sci Sports Exerc 33:1304–1310 Talbot LA, Gaines JM, Ling SM, Metter EJ (2003) A home-based protocol of electrical muscle stimulation for quadriceps muscle strength in older adults with osteoarthritis of the knee. J Rheumatol 30(7):1571–1578 Watson T (2000) The role of electrotherapy in contemporary physiotherapy practice. Man Ther 5(3):132–141 123