Participants
Eleven healthy males (height: 173.6 ± 7.53 cm; weight: 74.5 ± 17.3 kg; age: 24.6 ± 4.76 y) with no reported lower body injury 2 months prior to experimentation were recruited by YSC for this pilot study. Participants were asked to avoid any strenuous physical activity and caffeine containing food/beverage 24 hours prior to experimentation. Last meal intake was at least 2 hours before testing. Sample size in this pilot study was based on the study of Oza and colleagues [28]. A written informed consent was collected before any further experimentation. Ethical clearance for this study was obtained from the University of Tasmania Human Research Ethics Committee (H0016508), in agreement with the University of Taipei.
Procedures
The participants attended three experimentation sessions, separated by 48 h, at the Exercise Performance Laboratory of University of Taipei. Measurement of anthropometrics (height and weight), identification of RF, and familiarization of MVC protocol were administered in the first session. In the second session, a randomized 10 min HRV BFB or CON was facilitated. Randomization was carried out by JCP using an online random allocation tool [29]. Upon arrival at the testing facility, an electrode attachment to the soleus muscle (SOL) for surface electromyography (EMG) and stimulation electrodes on popliteal fossa and over the patella of the right limb were applied. This was followed by pre-testing for H-reflex. Subsequently, the participants performed HRV BFB/CON with knee angle set at approximately 90-120 degrees. HRV BFB was carried out using a commercial HRV BFB equipment (HRV Starter System, Thought Technology, Canada). Participants under CON seated quietly and performed normal breathing for 10 min while asked to gaze at the computer screen. HRV BFB/CON was succeeded by post H-reflex test. Then, participants performed MVC testing. A crossover intervention was performed on Day 3. Figure 1 displays the experimental protocol for this study.
Resonance frequency
An elastic belt and finger sensor were placed on the participant to allow simultaneous recording of respiration and heart rate (HRV Starter System, Thought Technology, Canada). Then, a 5-minute familiarization of diaphragmatic breathing with visual feedback occurred. This was followed by RF testing that involves two-minute breathing at various frequencies (6.5, 6.0, 5.5, 5.0, 4.5 times per min). RF is established as the breathing pace that displayed the highest amplitude in the low frequency of HRV [5, 30].
H –reflex
H-reflex was measured from the soleus (SOL) electromyography (EMG). A surface electrode (TSD150A, Biopac Systems, USA) was placed 2/3 from the medial condyles of the femur to the medial malleolus and central position in the medial-lateral direction of the border of SOL. In addition, a reference electrode was attached on the right hand of the participants. Further, an adhesive electrode (10 x 10 mm, FA 25, Gem-Stick, Australia) was placed on the right popliteal fossa as cathode and another (50 x 50 mm, Life Care, Taiwan) adhesive electrode was fixed over the right patella as the anode. After, participants were asked to seat with right knee extended at approximately 180 degrees, while the left knee flexed at 90-120 degrees. Hip flexion was 90-120 degrees. Recording of EMG signals were acquired using a commercial data acquisition system (MP150, Biopac Systems, USA) filtered with a band-pass range of 10-500 Hz and amplified with a gain of 1,000 times. The common mode rejection ratio for the EMG amplifier was 100dB. Sampling rate was set to 2.5 kHz. A single electrical impulse was applied to the right posterior tibial nerve via an electrical stimulator (DSH7, Digitimer, Herfordshire, UK) with 1000µs pulse duration to identify SOL H-reflex response. To determine maximal levels of H-wave and M-wave, stimulation intensity was increased with 10 mA increments until maximum M-wave (Mmax) was identified. The stimulation intensity for exhibiting maximum H-wave (Hmax) was determined by using 2 mA increment when the H-reflex threshold was identified (see Figure 2). To record the H-reflex responses for comparison, stimulation intensity was controlled to elicit an H-reflex response with a small incidence of M wave at 10% to 20% Mmax [31]. Each stimulation intensity was applied four times with 10 s inter stimulation interval. A commercial data acquisition system (Acqknowledge 4.2.1 software, Biopac Inc., USA) with custom-written program was used to synchronize the electrical stimulation trigger and EMG recording. H-reflex parameters that were utilised for analysis included M wave at maximal H-reflex (M wave at Hmax), Hmax, maximal M-reflex response (Mmax), Hmax/Mmax ratio (maximal Hmax response to normalized Mmax), and M wave at Hmax/Mmax ratio (M wave during Hmax normalized to maximal M-reflex response). Hmax estimates the number of activated Іa afferents elicited by electrical stimulations [22, 23]. Mmax represents the entire activation of descending inputs to motor axons [24, 30, 31]. Hmax/Mmax ratio is the proportion of the entire motoneuron pool capable of being recruited by Іa afferents [24, 32]. M wave at Hmax refers to the proportion of motor fibers activated by electrical stimulations [23, 31]. During H-reflex testing, participants were encouraged to breathe normally. A sample of H wave and M wave recruitment curves from one representative participant is displayed on Figure 2.
Maximal voluntary isometric contraction
An isokinetic dynamometer (Biodex System 4, Shirley, New York) was utilized to measure MVC of plantar flexors. In this test, participants performed three x 3-second plantar flexor MVC. Prior to MVC testing, the participants performed plantarflexion-dorsiflexion at force intensity of submaximal level as warm-up for 30 times, followed by a 4-minute rest. A visual feedback of torque production via a computer monitor located in front of the participant, and standardized verbal encouragement were provided to facilitate maximal effort during each trial. Rest interval between trials was one-minute. The highest MVC torque value was recorded for analysis.
Statistical Analyses
Data are expressed as mean ± standard deviation. Normality of data was analysed using Kolmogorov-Smirnov method. A 2 x 2 repeated measures ANOVA was employed to analyse any significant main effects (condition and time) and interaction (condition x time). Effect size was estimated using eta squared. Paired T-test was carried out for any significant parameter main effect. The same method was also used to establish any difference in MVC between HRV BFB and CON. Statistical procedures were carried out using a commercial statistical package (SPSS ver 22, IBM Corp, USA). Significance was set at alpha of 0.05.