Thirty-one healthy young adults (15 men and 16 women) participated in this cross-sectional study of repeated measures. Participants were recruited from a community health centre in Malaga (Spain) through ads placed in the centre. Data were collected in a human movement analysis laboratory between April 2014 and December 2014. The participants’ average (mean ± standard deviation) age, height, and body weight were 26.9 ± 5.7 years, 1.74 ± 0.12 m and 68.04 ± 12.54 kg, respectively. Exclusion criteria for the study included participants aged less than 18 or more than 40 years, neuro-musculoskeletal limitations that prevent completion of the protocol, cognitive impairment from any aetiology, surgical intervention in the lower limbs over the past 12 months (prior to the recruitment), BMI ≥ 35 kg/m2, severe metabolic, cardiovascular or respiratory problem and current pregnancy. Prior to the study, each participant provided written informed consent. The ethics committee of the Faculty of Health Sciences granted ethical approval for the present study. The study complied with the principles laid out in the Declaration of Helsinki. The participants’ personal data were treated in accordance with the Spanish Organic Law of Protection for Personal Data 15/1999.
Participants’ position
All participants sat with knees and hips flexed at 90°. From this position, each participant positioned the dominant foot on a device comprised of two platforms (one horizontal and one vertical) that allowed the foot to be placed in a neutral position (90° angle between the foot and leg) (Fig. 1). Velcro® straps were used to prevent leg and foot movement during the execution of IFD.
Torque measurement
A load cell (Biomotor Mega 6000 accessories – Mega Electronics Ltd, Kuopio, Finland) [18] was positioned, connected by chains, between the horizontal platform and the ground (Fig. 1). To ensure the IFD, the load cell blocked the movement during FD. The torque measurement was performed throughout the duration of the IFD (five seconds). The maximum value measured during IFD was considered the maximum torque and was used to normalise the variables and analyse offline SMG and EMG variables.
Ultrasound measurement
The Esaote MyLab25 Gold ultrasound system was used to acquire ultrasound. A probe model LA523 [19], with a frequency of 12 Hz at 5 cm deep, was used to acquire ultrasound images. An operator with extensive experience in musculoskeletal SMG performed the ultrasound image acquisition. The probe was placed on the right leg on the first third of the leg, parallel to the major axis of the TA muscle, using the tibial tuberosity as a reference (the probe was placed below) [13]. Before the study, extensive pilot testing with reference to anatomy textbooks, models and SMG studies [1,203] was conducted to determine the best scanning protocol.
Electromyography measurement
Activation of the TA muscle was measured using the Biomonitor ME6000 electromyograph (Mega Electronics Ltd, Kuopio, Finland) [18] with a sampling frequency of 1000 Hz. The electrodes were positioned and the skin prepared in accordance with the European Recommendations for Surface Electromyography (sEMG) [2, 20, 21]. In order to avoid EMG crosstalk, special attention was paid to the placement of the electrodes, as well as to the spacing and size [2, 20]. A space was provided for placing the ultrasound probe without affecting the position and operation of the electrodes. The line between the medial malleolus and the fibula’s head was used to position the electrodes. After a TA muscle belly palpation, the electrodes were positioned in the proximal third (the reference line), with a distance between the electrodes of 2 cm. Following the electromyograph manufacturer’s protocol (Mega Electronics Ltd, Kuopio, Finland) [18]) for each participant, three Al/AgCl electrodes (5 cm in diameter) were used (two electrodes were used as poles, while the third electrode was used as a reference). To remove high-frequency noise, the signal was filtered via a low pass Butterworth filter (bidirectional fourth-order).
Synchronisation data acquisition
The Megawin 3.0.1 software and a Biomonitor ME6000 console [Mega Electronics Ltd] [18] connected to each device were used to continuously and synchronously record the SMG and sEMG data. Torque was the reference variable used in order to subsequently measure SMG variables (thickness and pennation angle) and EMG variables of the TA. The start and end of the synchronising of all systems during each test were controlled using a trigger device [Mega Electronics] [18].
Experimental procedure
For all participants, the measurements were performed on the dominant foot. Before the experimental procedure, each participant was able to perform all repetitions deemed necessary to become familiar with the protocol. Three maximal IFD (defined as the highest torque value measured [11]) were carried out for five seconds (The rest between each repetition was 90 s to prevent that fatigue that could influence contractions). Torque, SMG and EMG signals were collected during the test.
Relative contractions
The torque value recorded during maximal voluntary contraction (MVC) was used as a reference for calculating the intensity of the MVC relative contractions. Participants performed 75, 50 and 25 % of their torque recorded during MVC. Each participant performed three repetitions for each IFD relative contraction, with 90 s of rest between each repetition. All participants performed the same sequence of IFD (100 % – 75 % – 50 % – 25 % MVC). Torque, SMG (pennation angle and muscle thickness) and EMG variables (EMGAreaUnderCurve (EMGAUC) and EMGMaximumPeak (EMGMP)) were measured offline from the data collected during the protocol.
Data analysis
The instant at which maximum torque was recorded was used as a reference for extracting the EMG and SMG variables. Architectural variables (thickness and angle pennation muscle) were calculated offline. The ultrasound pictures were imported into a specific programme for the processing and analysis of images (AutoCAD 2012 – English SP2 software (Autodesk, San Rafael, California, USA)). Pennation angle was considered as the distance between the central intramuscular septum and the line of the clearest fascicle was considered as the positive angle [9]. In addition, muscle thickness was considered as the distance between the superficial and deep muscle aponeuroses [9].
The software MegaWin 3.0.1 (Mega Electronics Ltd, Kuopio, Finland) [18] was used to record and process the EMG signal. The muscle functional variables (EMGMP and EMGAUC) were extracted from the raw results of a selected area (encompasses the EMGMP and one second before and after this time point). An independent and blinded researcher analysed the data (Fig. 2).
Statistical analysis
The reliability (considered as a test–retest standard deviation of differences as the 95 % limits of agreement) was calculated for each outcome variable (torque, EMGAUC, EMGMP, muscle thickness and pennation angle). For this purpose, the three measures were acquired during each IFD intensity and were used to calculate the standard error of the measurement (SEM) and the internal consistency (Cronbach’s alpha) of the measure, together with the 95 % confidence interval for each variable. Reliability was classified as follows: excellent (Cronbach’s alpha: > 0.80), good (Cronbach’s alpha: 0.80–0.60), moderate (Cronbach’s alpha 0.60–0 .40), or poor (Cronbach’s alpha <0.40) [22, 23].
A descriptive statistical analysis was carried out using mean and standard deviation. An inferential analysis was carried out using a Pearson correlations(r) in accordance with the normality of the variables after a sample K–S test. For each contraction intensity, an exponential regression analysis was performed, where the dependent variable was torque and the independent variables were EMGAUC, EMGMP, muscle thickness and pennation angle. The magnitude of the correlations was as follows: strong (r > 0.75), moderate (0.50–0.74), or poor (r < 0.49) [24].
The software G Power (Version 3.1) was used to estimate the sample size. In the a priori calculation, based on the literature [13], a minimum of 26 subjects was necessary to have sufficient statistical power (80 %) and an alpha error of 0.05. The statistical analysis was carried out using the SPSS 21.0 statistical package for Windows.