Experimental design and participants
The participants visited our laboratory on four separate days. On the first day, measurement of the load of 10 RM of parallel squat and familiarization with the strength measurement and squat exercises were conducted. On the second to fourth days, the participants joined the following three interventions during quiet sitting for 30 min in random order: tonic vibration to the right thigh’s VL muscle belly (VL condition), tonic vibration to the right thigh’s biceps femoris long head (BF) muscle belly (BF condition) and quiet sitting without any vibration (CON condition). We selected VL and BF as target muscles since they have relatively large physiological cross-sectional areas (Ward et al. 2009) and muscle volumes (Ema et al. 2016b) among the constituents of the quadriceps femoris and hamstrings, respectively. Before and after the interventions, measurements of maximal isometric knee extension and flexion strength and parallel squat at 10 RM were performed. All measurements were completed within 5 min after the intervention, so the reduction of Ia afferents activity following prolonged vibration for 30 min would still be in effect (Thompson and Bélanger 2002). We performed a priori sample size estimation (G*Power 3.1.7, Kiel University, Germany) to detect a significant change in knee extension torque following VL vibration for 30 min by a paired t test, using α = 0.05, power at 0.80, and data of our pilot study [n = 11, mean of difference and standard deviation (SD) of difference in MVCKE torque before and after the vibration were 13 and 10 Nm, respectively]. The estimation demonstrated that six participants would be needed to find the expected change. In the current study, fourteen untrained healthy men (age, 22 ± 2 year; height, 1.72 ± 0.04 m; body mass, 64 ± 6 kg; mean ± SD) who had no injuries of the lower extremity participated. Prior to the execution of the experiments, the participants were informed of the purpose and risks of the study and provided written informed consent. This study was approved by the Ethics Committee of the Shibaura Institute of Technology.
Electromyography (EMG) measurements
Surface EMG signals were recorded from VL, VM, RF, BF and semimembranosus (SM) using Ag/AgCl electrodes (BlueSensor N-00-S, Ambu A/S, Denmark) with an interelectrode distance of 20 mm. The electrodes were placed at the level of 90% (VM), 70% (SM), 50% (VL and BF) and 40% (RF) of the thigh length which was determined as the distance from greater trochanter to popliteal crease, after the identification of muscle belly and fascicle directions using B-mode ultrasonography (ACUSON S2000, Siemens Medical Solutions, USA) so as to reduce the effect of cross talk. The electrode placement was preceded by abrasion of the skin surface to reduce the source impedance to less than 5 kΩ. The EMG signals were high-pass filtered (5 Hz) and amplified (MEG-6108, Nihon Koden, Japan). The reference electrode was placed on the right patella for all EMG measurements. To match the electrode placement among the three different conditions, the participants were requested to maintain some pen marks that indicated the electrode placements on the skin throughout the experiments.
The participant sat in a specially customized dynamometer (Hamano Seisakusho, Japan) and remained relaxed during the interventions. The knee and hip joint angles were 75° and 80° (anatomical position = 0˚), respectively. In the vibration conditions, tonic vibration was applied for 30 min perpendicular to right VL (i.e., from right side of the thigh) and BF (i.e., from back side of the thigh) slightly proximal top the region of EMG electrodes using a vibration generator (WaveMaker05, Asahi Seisakusyo, Japan). To selectively activate Ia afferents, the vibration frequency was set at 80 Hz (Roll et al. 1989). The force of the vibration was measured using a load cell (LUR-A50NSA1, Kyowa, Japan) attached to the vibration generator. The forces before and during the vibration and peak-to-peak amplitude of the vibration were controlled at 7 N, 10–15 N and 1.6 mm, respectively. They were similar to those of the previous study that indicated a significant reduction of Ia afferent activity accompanied by the corresponding decrease in MVC torque and agonist muscle activations after 30 min vibration (Ushiyama et al. 2005).
Before and after the intervention for 30 min, isometric knee extension and flexion strengths with maximal effort were measured (Fig. 1). The participant sat on the bench of the dynamometer with their pelvis secured to the bench by a non-elastic strap. Care was taken to adjust the centers of rotation of the dynamometer and knee joint. The knee and hip joint angles were consistent with those during tonic vibration. Before each intervention, the participant was asked to extend or flex the knee twice with maximal effort. If the difference in peak value between the two contractions was above 10%, a third trial was allowed. Immediately after the 30 min intervention, the knee extension and flexion strength trials were again performed once each. The peak torque was defined as MVC torque (knee extension, MVCKE; knee flexion, MVCKF).
The free-weight parallel squat was performed before and after the intervention. To match the joint kinematics before and after the intervention and among the three conditions, following procedures were performed. The participant was instructed to stand equally on both legs on the floor with their feet shoulder-width apart and angled outward at approximately 30°. The standing position relative to the experimental setup was fixed in each participant throughout the experiment. From the standing posture, the participant performed parallel squats at 10 RM load (one set of five repetitions), consisting of lengthening action (2 s) and shortening action (2 s), with the aid of a metronome. The knee joint angle was measured using an electronic goniometer (SG150, Biometrics, UK). Parallel squat depth was defined in advance at the position at which the thigh was parallel to the floor. The depth was controlled using a tense rope set at the height of each participant’s squat depth and feedback was provided by an examiner throughout the exercises. As a result, a two-way analysis of variance (ANOVA) indicated that there was no main effect of time (before and after the intervention, P = 0.250) or condition (VL, BF and CON conditions, P = 0.570) or interaction of the two factors (P = 0.162) on knee joint angle at the depth position. This suggests that knee and hip joint kinematics during the squat exercise was almost matched throughout the experiment by controlling of the standing position and squat depth. The determination of 10 RM load of the parallel squat was performed after several submaximal squat exercises at light-to-moderate load as a warm-up. The load was then increased until the participant could successfully lower and raise the bar from a sitting position in which the thigh was parallel with the floor 10 times but failed to achieve an 11th repetition. The participant determined his 10 RM (59 ± 10 kg) with sufficient rest within four attempts.
The EMG, torque, force of vibration and knee joint angle data were simultaneously recorded at 1 kHz sampling frequency and stored in a personal computer after A/D conversion (PowerLab16/35, ADInstruments, Australia). In the strength measurements before the intervention, data during the two trials were averaged and used for further analyses. The root mean square values of EMG signals (RMS-EMGs) were calculated over a 0.5 s period around the peak torque. For the parallel squat, the RMS-EMG at each repetition was calculated separately in the lengthening and shortening phases, which were determined from the knee joint angle data, and data of five repetitions were averaged in each phase. Each muscle’s RMS-EMG was normalized to that during MVC trials before the intervention. In addition, to examine the inter-muscle difference in the magnitude of muscle activations, the RMS-EMGs were also averaged between lengthening and shortening phases before the interventions.
Data are presented as means ± SDs. The statistical analyses were performed using SPSS version 22 (IBM, USA). A two-way ANOVA with repeated measures was conducted to determine the effects of time (before and after the intervention) and condition (VL, BF and CON conditions) on MVC torques and RMS-EMG during MVC trials in each muscle. The relationship between relative change in RMS-EMG of the vibrated muscle and relative change in MVC torque was tested using Pearson’s product moment correlation coefficient. A two-way ANOVA with repeated measures was used to determine whether normalized RMS-EMGs (means of the lengthening and shortening phases) during the squat before the intervention differed among the muscles (VL, VM, RF, BF and SM) and three conditions. To examine the effects of time, condition and phase (lengthening and shortening), a three-way ANOVA with repeated measures was performed on normalized RMS-EMG during parallel squat exercises in each muscle. When a significant interaction or main effect of time was shown, following ANOVA with the Bonferroni multiple-comparison test was used to examine the differences in variables before and after the intervention in each condition and phase. The significance level was set at P < 0.05.