Eur J Appl Physiol (2006) 98:79–87

DOI 10.1007/s00421-006-0250-5

ORIGINAL ARTICLE

Motor performance changes induced by muscle vibration

Luigi Fattorini · Aldo Ferraresi · Angelo Rodio · Gian Battista Azzena · Guido Maria Filippi

Accepted: 24 May 2006 / Published online: 9 August 2006© Springer-Verlag 2006

Abstract The possibility that mechanical stimulationof selected muscles can act directly on the nervous sys-tem inducing persistent changes of motor perfor-mances was explored. On the basis of literature,stimulating parameters were chosen to stimulate thecentral nervous system and to avoid muscle Wbre inju-ries. A sinusoidal mechanical vibration was applied, forthree consecutive days, on the quadriceps muscle inseven subjects that performed a muscular contraction(VC). The same stimulation paradigm was applied onseven subjects in relaxed muscle condition (VR) andseven subjects were not treated at all (NV). Two ses-sions (PRE and POST) of isometric and isotonic testswere performed separated for 21 days, in all studiedgroups 7 days before and 15 days after stimulation,whilst an isokinetic test was performed on VC only. Inthe isometric test, the time of force developmentshowed a signiWcant decrease only in VC (POST vsPRE mean 27.8%, P < 0.05). In the isotonic test, thesubjects’ had to perform a fatiguing leg extensionagainst a load. In this condition, the fatigue resistanceincreased greatly in VC (mean 40.3%, P < 0.001),

increased slightly in VR and there was no diVerence inNV. In Isokinetic test, at several angular velocities, sig-niWcantly less time was required to reach the forcepeak (mean 20.2% P < 0.05). The Wndings could beascribed to plastic changes in proprioceptive process-ing, leading to an improvement in knee joint control.Such action delineates a new tool in sports training andin motor rehabilitation.

Keywords Fatigue · Force development · Proprioception · Functional stiVness · Muscle vibration

Introduction

Muscle vibration is a powerful tool for the stimulationof proprioceptors. A great deal of research has beendevoted to investigating its use in clinical therapy andsports training to enhance motor functions through animprovement in motor control. Even if some reports(Bosco et al. 1999a, b, 2000) have suggested positiveeVects resulting from whole body vibration (WBV),international literature claims that repetitive and long-lasting exposure to WBV can be very dangerous tohealth, producing persisting damage to tissues(Bovenzi 1998; Lings and Leboeuf-Yde 2000; Seidel2005). Tissue injuries can be due to the propagation ofmechanical waves from the position of application tothe whole body. Thus, the mechanical stimulus param-eters can be distorted (Matsumoto and GriYn 2002)and reaching frequency values that may become dan-gerous (Seidel 2005) for the main joints (i.e. knee, hip,shoulder and vertebral bodies).

Other than WBV, mechanical perturbations of smallamplitude and localized on a speciWc body segment can

L. Fattorini (&)Dept. of Human Physiology, University of Rome“La Sapienza”, P.le Aldo Moro 5, 00185 Rome, Italye-mail: luigi.fattorini@uniroma1.it

A. Ferraresi · G. B. Azzena · G. M. FilippiInstitute of Human Physiology, Università Cattolicadi Roma, L.go F. Vito 1, 00168 Rome, Italy

A. RodioDepartment of Motor Sciences and Health, University of Cassino, Viale Bonomi, 03043 Cassino (Fr), Italy

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easily be kept within safety ranges, below 100 �m(Necking et al. 1996). Positive therapeutic eVects havebeen reported in diVerent experimental conditions.Such eVects have been very short lasting, or vanished asthe stimulus ended (Cannon et al. 1987; Karnath et al.2000). Other studies have suggested that long-lastingeVects may be induced by a speciWc vibration frequencyand application time (Kawakami et al. 2001; Rosenk-ranz and Rothwell 2003, 2004; Steyvers et al. 2003). Theuse of a vibration of a narrow frequency range (80–120 Hz) applied locally (Rosenkranz and Rothwell2003) for about 10–15 min (Rosenkranz and Rothwell2004) can modify proprioceptive control for up to30 min after stopping stimulus. All reports agree inascribing such long-lasting eVects to the changes in aVer-ent–eVerent processing and not to modiWcations local-ized within the treated muscular apparatus (i.e. vascularor metabolic mechanisms). Moreover, some researchhas shown that plastic changes in the central nervoussystem depend on the combined eVects of the aVerentstimulus pattern and the subject’s arousal (Rosenkranzand Rothwell 2004; Recanzone et al. 1992a, b).

Recently, Brunetti et al. (2006) have reportedchanges in balance capacity after anterior cruciate liga-ment reconstruction in patients treated with a stimula-tion protocol and have shown important and stableeVects of between 10 and 180 days. Consequently, it canbe hypothesized that muscle vibration could induceimportant adaptive plastic changes in motor control.The aim of the present research was to evaluate theinXuence of localized muscle vibration associated to anarousal state on motor performances of healthy subjects

Materials and methods

Subjects

Twenty-one healthy adults, 15 males, 6 females, volun-teered to participate in the study (age 31 § 2.4 years;height 176 § 6.2 cm; weight 67.5 § 5.3 kg). The studywas approved by the local ethics committee and all indi-viduals gave informed consent before participating in thestudy. Care was taken to exclude the subjects, who par-ticipated in the experiment, with sensorimotor patholo-gies. All individuals reported a moderate level ofphysical activity but they were not involved in any kindof systematic training, especially not during this research.

Experimental protocol

Male and female subjects were divided into threegroups and each group was comprised of Wve males and

two females. The experimental protocol was devised asfollows. Session 1 (PRE): isometric, isokinetic and iso-tonic motor tests were performed on three separatedays before stimulation. Session 2: 14 subjects belong-ing to VC and VR underwent a conditioning protocolby means of mechanical vibrations on the left leg; thissession was carried out 7 days after the PRE. Session 3(POST): measurements of PRE were repeated after3 weeks. All measurements of this work were per-formed on both legs in random sequence. Below theconditioning protocol of session 2 and isometric, isoki-netic and isotonic tasks of PRE and POST aredescribed.

Conditioning protocol

Sequences of mechanical perturbation were applied onthree components of QF (quadriceps femoris) muscle:vastus medialis (VM), rectus femoris (RF) and inter-medius femoris (IF). Mechanical perturbation wasapplied by a commercial instrument (CRO®SYSTEM,NEMOCO srl, Italy). The instrument consisted of anelectromechanical transducer, a speciWc mechanicalsupport and an electronic control device. The mechani-cal support allowed the orientation, positioning andrigid Wxation of the transducer in every direction rela-tive to the individual’s body. The transducer was posi-tioned against the distal end of VM and the commontendon of RF and IF, at about 2 cm from the rotulamargin. The mechanical support allowed the compres-sion of soft tissues overlying the muscle–tendon com-plex with a static load of about 0.08–0.12 N in order tominimize superWcial tissue compliances. Support char-acteristics guaranteed an adequate rigidity of the sys-tem to transfer all the supplied force to the humantissue. The transducer was driven to producesequences of perturbations consisting of sinusoidalforces ranging between 7 and 9 N, at a Wxed frequencyof 100 Hz, the range of displacement was between0.005 and 0.015 mm peak to peak. During applicationof these force sequences, the subjects belonging to theVC group were invited to maintain an isometric con-traction on the treated muscle, by setting the contrac-tion level at about 20% of his maximum voluntarycontraction (MVC). The aim of this command was tofocus the subjects’ attention on the treated area and toenhance muscle stiVness, (Petit et al. 1990) reducingthe mechanical dumping of the applied perturbationsand allowing the mechanical forces a better propaga-tion within the muscle. Such sequences of micro-per-turbations were applied on three consecutive days,three times a day and each application lasted 10 min.The three consecutive applications were separated by a

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resting time of 30–60 s, during which the mechanicalapplication was interrupted and the subject relaxed themuscle.

Subjects belonging to VR were treated with thesame conditioning protocol of group VC, but the leftleg was relaxed during stimulation. An NV group didnot undergo vibration at all.

Isometric task

During both PRE and POST, measurements were per-formed separately in each leg to evaluate an isometricmuscular contraction on VC, VR and NV. Evaluationof the MVC developed by the QF was performed.MVC value was taken as the maximum of threeattempts, tests were separated one from the other by 5-min rest. During the test the subject was seated in acustomized chair (Fig. 1a), the vertical plane (P) of thechair was adjustable in rotation and translation to sethip (�) and knee angles (�) at 110 and 90° and capableof adapting to the individual’s anthropometric parame-ters. The subjects’ hips and shoulders were restrainedwith straps and the arms folded to avoid the possibilityof other muscles’ involvement in the movement. Aftera period of familiarization with the environment, ateach attempt the subject was encouraged to developthe maximal force as fast as possible. Subject wore aboot with a special orthosis that locked ankle joint at90( and a ring was placed on the rear side connectingthe piezoelectric force transducer (type 9311A, Kistler,Switzerland) to the chair. Force signal was ampliWedwith a charge ampliWer (type 5006, Kistler) and storedvia an A/D converter (ATMIO, 12 bit, sample fre-quency 2,048, National Instruments, USA) on a laptopcomputer.

Isokinetic task

Isokinetic task, separated by a day with respect to iso-metric task, consisted in the evaluation of the force–velocity relationship and was performed separately oneach leg on VC group. The maximal force exerted wasmeasured by a dynamometer (KIN COM Chatta-nooga, TN, USA) and subject’s positioning was setindividually, reproducing those used in isometric task.In order to do that, particular attention was paid toavoid the contribution of other muscles and thecounter-movements before the knee extension. Forcetrace was displayed on a personal computer screen toprovide participants with visual feedback. Angularvelocity values were Wxed at 30, 60, 90, 120, 180,240° s¡1 (range of motion was about 80°) and the sub-jects were requested to perform the movement as fastas possible. At each angular velocity the subject per-formed three contractions separated by 2 min of restbetween the various angular velocities, whilst rest timewas 10 min. The sequence of angular velocities wasdetermined at random.

Isotonic task

Isokinetic and isotonic tasks were separated by 1 day.Isotonic tasks were performed separately on each legand on all the three groups (VC, VR and NV) and con-sisted in leg exhaustive isotonic extensions. Subject waspositioned in the chair and wore the same orthosis as inisometric task and constrained at hip and knee level asdescribed previously. A load equal to 30% of the MVCwas used. This load was fastened to the end of an inelas-tic rope whilst the other side was fastened to the ortho-sis-heel. A series of Wxed pulleys allowed the load to belifted along a perpendicular trajectory to the Xoor(Fig. 1b). In each subject the load position (D) wasadjusted to avoid Xoor contact and the height was moni-tored by a graduated panel. The movement consisted ofa rapid maximum extension of the leg and in a con-trolled return to rest position. Movements were per-formed according to a frequency chosen by the subject;however, subjects were encouraged to adopt a consis-tent and regular frequency in all tests. The subject wasasked to begin a new extension when the previous onewas completely Wnished. Finally, motor task consisted ofthree series of repetitive movements until exhaustion.Sixty minutes of rest separated one series from another.Each series was considered Wnished when subject didnot start a new extension or when the altitude reachedby the load was less than 75% of its maximum exten-sion. The sum of movements performed in the threeseries was deWned as the index of isotonic performance.

Fig. 1 Experimental set up for isometric and isotonic tasks. aAdjustable vertical plane and relative angles at hip and knee. bArrangement of the pulleys and of the load. The load was adjust-ed to avoid Xoor contact

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Force development (FD)

During the MVC test, force signal was recorded andsubsequently analysed to compute the force levelreached in deWned time. The FD is deWned as the forcevalue reached at 30, 50, 100 and 200 ms after the onsettime. To avoid transient phenomenon the onset timewas deWned when force reached 5% of its maximum.To compare measurements among all subjects, theindividual’s FD values were calculated as a percentageof his/her own MVC.

Statistical analysis

Data are presented as group mean value (§ SD).POST versus PRE was analysed using Student’s t testfor paired samples (two-tailed, 0.05 level of signiW-cance).

Results

As reported in Materials and methods all measureswere evaluated 7 days before conditioning protocol(PRE) and 15 days after the conditioning protocolended (POST).

Isometric tests

A comparison between MVC measured in PRE andPOST did not reveal any signiWcant changes in eitherleg in any of the subjects participating to the experi-mental research, as shown in Table 1.

On the other hand, even if MVC did not changed,the analysis of FD showed that POST treatment theforce developed at 30, 50, 100 and 200 ms statisticallysigniWcant increases in the left leg of all the subjectsbelonging to the VC group (Fig. 2). Developed forceincreased in the left leg of the VC group + 36.4%(§ 12.5) after 30 ms (P = 0.0057), + 34% (§ 28.6) after50 ms (P = 0.035), + 26.6% (§ 15) after 100 ms (P =0.007) and + 14.1% (§ 12.5) after 200 ms (P = 0.048),showing a grand average of + 27.8 § 10%. None of theFD values obtained after conditioning protocol turnedout signiWcant in the right leg of VC group. VR and NV

groups did not show any signiWcant change in eitherleg, even if VR showed a small mean increase in theleft leg.

Isokinetic tests

Isokinetic tests were performed only on VC group.Conditioning protocol did not produce any signiWcantmodiWcation in the force–velocity relationship, or atany of the tested angular velocities, in left versusright. In the left leg, the time elapsed to reach themaximal peak force, i.e. peak time (PT), was signiW-cantly reduced at all tested velocities, except at240°s¡1. In Fig. 3 the time to peak relative to thepooled subjects’ data is reported for each angularvelocity, in left and right legs. The mean PT reductionin the left leg was of ¡20.2 § 2.9%, the valuesobtained at the diVerent velocities ranged between¡12.6 and ¡23.2%. As in the case of the results in iso-metric test, no changes in right leg performance wereobserved.

Isotonic tests

As detailed in Materials and methods, all subjects per-formed three series of exhaustive isotonic leg exten-sions against the load, kinematic parameters arereported in Table 2 and none of the values relative tothe execution time course showed diVerences betweenPOST and PRE.

As previously reported the index of performance inthis test was computed by summing the number ofmovements executed in the three series. This index inthe PRE, in all three groups no signiWcant diVerencesbetween right and left legs were evidenced. Same indexshowed a percentage increase in the left and right legseither for all the three groups when comparison thePOST versus PRE, as depicted in Fig. 4. As evidencedPOST versus PRE comparison, in VC, showed a sig-niWcant increase of performance in the left leg (meanvalue 40.3 § 16.9%, P = 0.0002) and a smaller one inthe right leg (mean value 15.6 § 9.3%, P = 0.04). Asobserved in isometric tests, VC and NV group did notreveal any diVerences between PRE and POST in bothlegs.

Table 1 MVC values PRE and POST treatment

Mean values of MVC recorded in the three groups, PRE and POST treatments

MVC Group Left leg PRE Left leg POST Right leg PRE Right leg POST

Force (N) VC 429.8 § 98 450.7 § 100.3 448.2 § 81.8 480.4 § 100.7VR 434.5 § 91.5 479.2 § 99.2 450.7 § 82.3 461.2 § 83.2NV 446 § 98.3 458.4 § 97.6 439.8 § 96.1 457.6 § 99

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Discussion

Mechanical vibration is usually applied in two forms:one able to invade the whole body and another

localized and limited to well-deWned muscle area.International literature claims that the WBV is highlydangerous for bones (Bovenzi 1998; Lings and Leb-oeuf-Yde 2000), joints and muscles, even though some

Fig. 2 PRE and POST mean values of force, normalized to the MVC, evaluated at 30, 50, 100 and 200 ms from the iso-metric contraction onset. **P < 0.01, *P < 0.05

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authors have reported beneWts (Bosco et al. 1999a, b,2000). On the other hand, localized muscle vibrationhas shown various positive eVects on diVerent musclediseases (Cannon et al. 1987; Karnath et al. 2000; Wol-paw and Tennissen 2001). Generally, beneWts havebeen short lasting, even though, as indicated in theintroduction, on the basis of magnetic stimulation, ithas recently been shown that eVects following a stimu-lation protocol are present in sensory and sensorimotorcortical circuits for up to 30 min (Kawakami et al. 2001;Rosenkranz and Rothwell 2003, 2004). Recanzoneet al. (1992a, b) have demonstrated an eVect lastingmore than 24 h after stopping the stimulation. SucheVects are ascribed to plastic changes induced in thenervous system and the importance of the cortical par-ticipation is demonstrated by the fact that this eVect ispresent only if the subject’s attention is focused on thetreated area.

The aim of this research was to ascertain whethersequences of local mechanical micro-perturbationswith a safety displacement range (less than 20 �m)associated with light muscular contraction could inducepersisting modiWcations of the motor performance onhealthy subjects.

Results show that periodic mechanical stimuli, uni-laterally applied on RF, IF and VM muscles, while thesubject maintains an isometric contraction of thesame muscle (VC), can induce long-lasting eVects. Instimulated leg, 2 weeks after conditioning protocolended, the following results were observed: (1) in iso-metric conditions, an FD enhancement at 30, 50, 100,200 ms (mean + 27.8 § 10%), was associated with aninvariance of MVC; (2) in isokinetic conditions, areduction in PT (mean ¡20.2 § 2.9%), withoutchanging the force–velocity relationship; (3) in iso-tonic conditions, the subjects enhanced the number ofthe loaded leg extension movements (mean value40.3 § 16.9%). In the untreated right leg of VC onlythe changed parameter (number of loaded leg exten-sion movements) was detected in isotonic conditions(+ 15.6 § 9.3%). No statistically signiWcant eVectswere detected when subjects were treated in theabsence of voluntary contraction (VR) and in non-treated subjects (NV).

The absence of results in VR allows the exclusionof a placebo eVect in VC. Muscular hypertrophy canbe excluded, since 15 days are a too short an intervalto induce an increase in the muscle mass (Ahtiainenet al. 2003); furthermore, the fact that MVC andforce–velocity relations are unchanged is in accor-dance with an invariance in the contractile mass. Theabsence of any increase in MVC seems to excludeenhancements of motor units recruitment and also ahigher synchronization of motor units. Finally 15 daysof persisting FD changes, as well as the changes in thetime to peak and endurance in isotonic tests suggestplastic modiWcations to the nervous motor control.

Fig. 3 PRE and POST mean values of PT measured in isokinetictest. All left leg measurements showed a signiWcant diVerence.*P < 0.05

Table 2 Kinematic parameters of exhaustive isotonic leg extensions against the load

Mean values, among series and among subjects, of the leg extension parameters of the isotonic test. In table the reported are the num-bers of leg extension per second (frequency), duration of movement (execution time) and duration of resting phase i.e. the time intervalbetween two consecutive extensions (resting time)

Movement characteristics Group Left leg PRE Left leg POST Right leg PRE Right leg POST

Frequency (movements s¡1) VC 0.58 § 0.19 0.59 § 0.12 0.6 § 0.21 0.61 § 0.1VR 0.63 § 0.2 0.61 § 0.09 0.62 § 0.18 0.59 § 0.21NV 0.61 § 0.18 0.6 § 0.16 0.57 § 0.26 0.59 § 0.17

Execution time (s) VC 1.66 § 0.4 1.64 § 0.3 1.45 § 0.2 1.4 § 0.2VR 1.63 § 0.3 1.66 § 0.3 1.5 § 0.4 1.48 § 0.3NV 1.64 § 0.5 1.65 § 0.4 1.48 § 0.4 1.45 § 0.3

Resting time (s) VC 1.69 § 0.5 1.65 § 0.3 1.72 § 0.3 1.73 § 0.4VR 1.67 § 0.7 1.64 § 0.4 1.7 § 0.5 1.71 § 0.5NV 1.68 § 0.6 1.66 § 0.5 1.73 § 0.4 1.72 § 0.4

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FD changes suggest improved knee joint stabilizationdue to an improvement in the processing of proprio-ceptive information (Brunetti et al. 2006), which candetermine a diVerent motor control (Gruber and Gol-lhofer 2004).

The reported FD increase was conWrmed andexpanded in isokinetic tasks, where there was animportant and statistically signiWcant reduction in thePT, at all tested velocities (except 240°s¡1). Also inisokinetic tasks, time force development changes wereobtained without altering the maximal force. Thisresult suggests that not only the early phase of muscleactivation, but the whole movement is controlleddiVerently after conditioning protocol, supporting achange in proprioceptive information processing fol-lowing the conditioning protocol.

An improved proprioceptive motor control alsoseems to be supported by data collected in fatiguingisotonic leg extension movements. In this task all sub-jects increased the number of movements signiW-cantly. Since the subjects were asked to continue theexercise as long as possible (exhaustive task), theincrease in the number of repetitions may be consid-ered as an increase in their resistance to fatigue.Actually such an increase in fatigue resistance mightbe the behavioural expression of an improved controlof the knee’s functional stiVness, resulting fromimproved proprioceptive processing. Indeed, in ourexperimental set up an increased QF stiVness controlcould reduce the muscle Wbre lesions (Morgan et al.2004) during the eccentric contraction (in the con-trolled return of the leg at the rest position) thusreducing fatigue perception.

Since the eVects lasted over 15 days after treat-ment ending, some speculations on the possibleinvolved mechanisms can be discussed. A plastic

change in nervous networks can be supposed sinceproprioceptive circuits are a plastic substratum onwhich it is possible to act with diVerent form of con-ditioning paradigms, based on proprioceptive stimu-lation (Recanzone et al. 1992a, b; Rosenkranz andRothwell 2003, 2004; Wolpaw and Tennissen 2001).In fact, short or long-term (i.e. minutes or months)plastic changes of nervous circuits (Wolpaw 1997)can be induced by simple or complex combinationsof sensory inputs. Applied vibration frequency(100 Hz) is reported to be adequate to induce persist-ing eVects in nervous circuits (Rosenkranz and Roth-well 2003, 2004). Exposure time to stimulation is alsoan important factor (Wolpaw and Tennissen 2001)and, in our experimental set up, the 30 min per dayexposure for three consecutive days to mechanicalperturbations might constitute an adequate condi-tion to develop a long-term enhancement in nervouscircuitry. Moreover diVerent papers suggest that it ispossible to produce plastic changes in central ner-vous circuitry by combining the subject’s attentionwith diVerent forms of vibratory stimulation(Recanzone et al. 1992a, b) and that such protocolscan improve the sensorial discrimination (Recanzoneet al. 1992b). In this research eVects were onlyobtained in the VC, when the conditioning protocolwas applied in VR group, in which the vibration wasapplied in “passive” conditions, there were no signiW-cant eVects. On the other hand, in a recent publica-tion (Brunetti et al. 2006) it was evidenced that onlyby coupling the subject’s attention (muscle contrac-tion) and the muscle vibration was proprioceptiveknee control largely improved and the authors sug-gested that this was due to the development of anassociative conditioning paradigm. Even if an experi-mental set up cannot allow the individualising of theaction mechanisms and levels, it is possible to discussthe receptor type involved by the used stimulus.Muscle vibration is considered a powerful stimulusfor Golgi and spindle aVerents (Bianconi and VanDer Meulen 1963). Moreover, the applied frequencyis considered optimal for primary spindle aVerents(Roll et al. 1989). However, the very low values ofthe applied vibration displacement introduce thequestion on whether primary muscle spindle aVerentscan be excited. A classic paper on cat (Brown et al.1965) showed that some primary endings, during fusi-motor activation, could be activated by a vibratorydisplacement as low as 10 �m. Also in our experi-mental conditions, the presence of a voluntary con-traction enhanced fusimotor action and spindlesensitivity; however, the large quadriceps mass mightdump muscle vibration. It follows that our experi-

Fig. 4 POST versus PRE percentage increase of number of legextensions in isotonic condition in both legs. **P < 0.01, *P < 0.05

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mental set up may not be discriminative on thereceptor type involved and also the other mechano-receptor types have to be considered (Pacinianorgans, type III and IV endings), even if primaryspindle activation is a suggestive and immediatehypothesis. Finally, some speculations on the level ofconditioning action within the central nervous sys-tem can be considered: diVerent authors (Wolpawand Tennissen 2001) suggested that diVerent levels ofproprioceptive circuitry can be modulated by condi-tioning procedures. It was also evidenced that long-lasting muscle vibration, stimulating primary spindleaVerents, can induce persisting changes in motor cor-tex function (Siggelkow et al. 1999; Rosenkranz andRothwell 2003, 2004). Other authors reported thatMV could act bilaterally on cerebral cortex (Kossevet al. 2001): a bilateral action of MV, following uni-lateral treatment, might explain the observed crosseVect, following treatment on fatigue resistance, inisotonic task.

Conclusions

In this research it was evidenced that through particu-lar conditioning applied protocol it is possible toinduce a persisting motor performance improvement,otherwise only obtainable after many weeks of speciWcphysical training programs not only in healthy subjectsbut also in rehabilitation programs (Brunetti et al.2006). Moreover, presented data show that the proto-col can enhance FD and fatigue resistance.

Aknowledgments The authors are grateful to Dr Ilenia Bazzuc-chi for her technical and critical support, to Prof. Marco March-etti and Prof. Cesare Iani for their useful discussion andcomments on the manuscript. The authors are also indebted withNEMOCO srl for its technical supply.

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