NTMS was performed with an NBS system (version 4.3.1, Nexstim Plc, Helsinki, Finland) using a figure-of-eight coil with biphasic pulse waveform. The stimuli were targeted to the primary motor cortex of the left hemisphere. TMS-induced motor evoked potentials (MEPs) were recorded from contralateral peripheral muscles by integrated stimulus-locked electromyography (EMG). The targeted muscles were the first dorsal interosseus (FDI) when stimulating the representation area of the hand and the abductor hallucis brevis (AHB) when stimulating the representation area of the foot. Other muscles (abductor pollicis brevis, extensor carpi radialis, tibialis anterior and vastus lateralis) were monitored with EMG to minimize simultaneous muscle activation. The approval limit of MEPs was 50 µV (peak-to-peak amplitude) and all responses with preceding or concurrent muscle activation 50 ms before the stimulus were excluded from the analysis [
23].
The nTMS protocol was executed in accordance with International Federation of Clinical Neurophysiology guidelines [
12]. The hotspots for FDI and AHB, producing the MEPs with the highest amplitudes, were mapped from the primary motor cortex as previously described [
10]. An integrated aiming tool was used to rotate the coil at the hotspot in order to determine the optimal orientation. The cortical representation areas for FDI and AHB were mapped starting from the hotspots with the aid of a virtual grid (5 mm × 5 mm per square) and a stimulator intensity 105% of RMT [
24]. Two stimulations with approximately 5 s interval were applied to each square of the grid until no MEPs were induced at the outer borders of the formed area. The representation areas were calculated using the spline interpolation method [
24,
25]. The centers-of-gravity (CoGs) [
26] for motor representations were determined using the MRI coordinate space in the navigation system, which has the origin in the right-posterior-inferior corner of the image. Corticospinal excitability was evaluated by determining the resting motor threshold (RMT) using an integrated iterative threshold assessment tool and reported as stimulator output percentage (% of maximum stimulator output, %-MSO) as well as calculated electric field (EF) strength (V/m) [
13,
27,
28]. Corticospinal inhibition was evaluated by the silent period (SP), which is a stimulation induced break in voluntary muscle activation [
29]. SPs were elicited at 120% of the RMT [
30] and determined as an average of five trials. The Input–Output (IO) curve was determined from a sequence of 160 pulses (intensities from 80 to 150% of RMT in 10% steps and intervals from 3 to 5 s) in a randomized order. A Boltzmann sigmoidal function was used to determine the maximum value (IO-max), the mid-point of the curve (V50) and the slope of the curve (IO-slope) [
10,
31,
32]. The ability of the cortex to habituate to repetitive stimuli was assessed by repetition suppression (RS) paradigm, in which 120 stimuli were delivered in trains of four stimuli at 120% of RMT with stimulation interval of 1 s and trains repeated every 20 s. [
33]. The four MEP amplitudes within the RS trials were averaged over all trials to represent RS response with second to fourth MEP in the trials representing the suppressed level of RS [
34].