Participants
Fifteen healthy male subjects (semi-professional football players) from various local football teams were recruited for this study. The subjects were male and right leg dominant (age 24.4 ± 3.2 years; height 181.1 ± 6.4 cm; body mass 80.5 ± 11.6 kg; mean ± SD). Subjects suffering from any lower limb injury within the previous three months of the assessment were excluded from the study. No participants had any previous history of neuromuscular disorder and each gave written informed consent prior to the experiment. Ethical approval for the study was gained according to the guidelines of University of Roehampton.
Experimental procedure
Before the test, each subject carried out a 10-minute warm-up including static stretching of the quadriceps muscles, free standing squats and jogging. Knee extension torque was measured isometrically using the Cybex dynamometer with HUMAC Norm software (Cybex Norm Testing & Rehabilitation System, Cybex Norm Inc., Ronkonkoma, USA). The subject was then seated in the adjustable chair and was secured with safety straps around the waist and across the chest to limit any changes in position during testing. The lateral condyle of the knee joint was visually aligned with the centre of the rotational axis of the dynamometer. The ankle cuff on the dynamometer’s lever arm was placed 2 cm proximal to the lateral malleolus, this allowed subjects to exert maximal forces without receiving any discomfort at the shin. Knee extension contractions were performed at a knee flexion angle of 75° flexion. The right leg was chosen for measurements, which was the dominant leg in all subjects.
The location for electrodes placement was taken from the European Recommendations for Surface Electromyography (SENIAM 1999) and ground electrode was placed in the wrist of subject. The subject was marked up using a washable marker pen and a razor was used to remove any hair on the skin that was cleansed with alcohol wipes and dried. The electrode for the vastus lateralis was placed 2/3 on the line from the anterior spina iliaca superior to the lateral side of the patella. The electrode for the rectus femoris was placed at 1/2 on the line from the anterior spina iliaca superior to the superior part of the patella.
After several familiarization contractions, each subject was asked to extend the knee against the lever actuator of the dynamometer by increasing the contraction force little by little to the maximal contraction and then relaxing gradually. A real time visual reference for the maximum torque was displayed on the isokinetic dynamometer computer screen that assisted with targeting. The maximal torque value was considered as the maximal voluntary contractions (MVC). Two repeated voluntary contractions were performed continuously within each trial with a 5 minutes rest between trials. The assessment included two trials at rest, maximum voluntary contraction (MVC), 75%, 50% and 25% for the rectus femoris and vastus lateralis whilst simultaneously recording sEMG and US images with support of a trigger system. Once the test procedure was completed, a 5-minute cool down was undertaken by each subject. This consisted of slow walking and static stretching of the quadriceps muscles for a duration of 15–20 seconds.
Data acquisition
The Cybex dynamometer was used to measure the isometric peak torques at 75° of knee flexion. Verbal encouragement and feedback was given to each subject for each contraction. A value of ±5 Nm of the actual torque target was accepted and recorded. Once the subject reached the required torque target point for each increment, they were instructed to hold the contraction for five seconds before being told to relax.
EMG data was measured using Biometrics DataLink Software (Ladysmith, VA, USA). sEMG activity was recorded with the use of a data logger with the start of the recording taking place just before the contraction and stopped when the subject had finished contracting and was in a relaxed position.
The Ultrasound scan was measured using Sonosite M-turbo with a high frequency transducer type HFL38x (13–6 MHz) (Bothell, WA, USA). The same operator performed all measurements. The thickness of the rectus femoris was measured with the probe placed in the transverse plane, and the angles of pennation of the vastus lateralis with the probe placed in the sagittal plane. A transducer was used to record the ultrasound images and placed inferior to the surface electrode placements of the rectus femoris and vastus lateralis. Liberal amounts of conductive gel were applied to the skin and the probe to obtain a clear and good quality image.
An external trigger with sEMG synchronized the ultrasound images once the subject had met the torque target value and was holding the contraction for 5 seconds. Two trials were for each muscle, beginning with RF muscle. Resting periods between each trial for the subject lasted for 5 minutes before the second trial began.
Data analysis
The sEMG data were sampled at 1000 Hz, and the data for the two trials at rest, MVC, 75%, 50% and 25% were averaged for both muscles. The signals were further processed by low pass filtering the signal with a Butterwotrth filter at a cut-off frequency of 500 Hz. The root mean square (RMS) sEMG amplitude was measured for 5 seconds and was then normalized to the average from MVC, normalized sEMG was used as a measure of muscle activation during contraction. Normalized sEMG ranged between 0 and 1.0. Ultrasound images for the two trials at rest, MVC, 75%, 50% and 25% were averaged for both the rectus femoris and vastus lateralis muscles. The thickness was measured from the anterior aspect of rectus femoris to the fascia (Figure 1). The pennation angle was determined from the angle between the fascia and the muscle fibres (Figure 2). All measurements were taken using the ImageJ software (National Institutes of Health, Bethesda, Maryland).
Statistical analysis
Statistical analysis was performed using SPSS 15.0 statistical package for Windows. Linear regressions were used for each muscle with a p-value less than 0.05 considered statistically significant. EMG data were used alone as input and pennation angle and muscle thickness how output.