Ten healthy subjects (S1–S10, age 26.5 ± 2.1 years, one female, all right-handed) recruited from Zhengzhou University participated in the experiment. All of the participants had normal or corrected-to-normal vision. Prior to the experiment, they were informed of the experimental procedure and signed a letter of consent. The study was approved by the local ethics committee for the Protection of Human Subjects for the Zhengzhou University.

Each participant was seated in a comfortable chair in a room with normal lighting and temperature. The participant sat facing a screen and was asked to watch the center of the screen. During the recording, the participant was asked to try to avoid eye movement, swallowing, and unnecessary limb movements.

At the beginning of each trial, a white cross was presented in the center of the screen (Figure 1). For the next 3 s, participants remained idle, with their hands, forearms, and elbows resting on the armrest of the chair. Next, a green arrow pointing either left or right appeared in the center of the screen for 0.5 s. After the cue disappeared, the participants prepared to perform the corresponding task instructed by the visual cue (left hand movement for left-pointing arrow, right hand movement for right-pointing arrow). After a preparation time of about 2 s, the participants performed the hand movement. Then, five seconds after the visual cue, an auditory cue was presented to inform the participant to return to the idle state. Six sessions were conducted for each participant, with 40 trials per session (20 trials each for left and right).

Two bipolar electrodes were attached to the participant's left and right arm to record electromyograms (EMG) during arm movement.

EEG data was recorded using a Neuroscan NuAmps digital amplifier system with 58 electrodes arranged in the standard 10–20 EEG configuration. All of the brain regions were covered by these electrodes. Two extended bipolar channels (BP3 and BP4) were used to acquire the left and right arms' EMG signals. The EEG signals were acquired at a sampling rate of 1,000 Hz with the Cz-REF as a reference, and the impedance of all electrodes was less than 5 KΩ. The Cz-REF was located between the Cz and CPZ electrodes. The selected electrodes and the Cz-REF are shown in Figure 2.

Data analysis consisted of two parts: EMG analysis and EEG data analysis. We acquired the onset time of hand movement for each trial by processing the EMG data, as shown in Figure 3. The event-related potential, brain voltage topographies, and classification accuracy of the RP state and idle state under different references were acquired by processing the EEG data.

The EMG data were filtered by a basic finite impulse response filter with respective cutoff frequencies of 6 and 50 Hz. We then calculated the energy of the filtered data and set the proper threshold to detect the onset time of motion. We recorded the onset times in a TXT file for EEGLAB toolbox analysis (Delorme and Makeig, 2004).

For the EEG data, EOG artifacts were first removed by Scan 4.5 software (the threshold was set at 60 μV). Then the processed data was exported to EEGLAB for further analysis.

Both the CAR and REST (Yao, 2001, 2017) were conducted offline to generate the re-referenced EEG. The CAR was conducted using the reref function from the EEGLAB toolbox (Delorme and Makeig, 2004), and the REST was conducted using the rest_refer function (http://www.neuro.uestc.edu.cn/REST/).

The original and re-referenced EEG were filtered by a band-pass filter (basic FIR filter, 0.1–1 Hz) and segmented into epochs from −3.5 to −3.4 s with respect to the onset time of motor execution (0 s). The data in the interval [−3.5, −3.4 s] was considered the baseline, and baseline correction was conducted for the segmented EEG epoch.

In order to examine the influence of different reference electrodes on the latency, amplitude, and waveform of RP, the RPs were plotted for the three reference methods. Then, we measured the RP's peak amplitude and latency on channels Cz, C1, C2, FC1, FC2, FCz, F1, F2, and Fz under different reference conditions.

Brain voltage topographies can reflect the topological structure of brain activation. It was important for us to analyze the activation areas of the brain and their change over time. However, previous studies usually drew the RP topographies under only one reference method (such as Cz-REF, CAR, or REST) and ignored the influence of different references. In our study, the 0.5 s averaged EEG segments before motion onset were extracted and voltage topographies were drawn every 0.1 s under the three reference methods.

Since RP has obvious time-domain features, we performed classification by the time-domain features. First, the preprocessed data (re-referenced, filtered, segmented, and baseline corrected) were resampled to 10 Hz. Then, the data were divided into two datasets: an idle dataset (i.e., no hand movement) and an RP dataset (i.e., left or right hand movement preparation). We extracted [−2.8 s, −2.2 s] and [−0.6 s, 0 s] of the signals above as idle and RP datasets, respectively (Figure 4).

According to the activated brain regions of RP, the channels (including F3, FZ, F4, FC1, FC2, C3, C4, Cz, CP1, and CP2) were selected for feature extraction in order to ensure the classification accuracy of different subjects. Finally, 60 features were acquired for each trial.

The LIBSVM 3.11 toolbox was used for classification (Chih-Chung and Chih-Jen, 2011). The radial basis function kernel was utilized, and the penalty factor (C) and gamma (Γ) parameters were optimized by the grid method. The classification accuracy of the RP and idle state was obtained using 5-fold cross-validation.

Figure 5 shows S7's grand average of the RP interval in three references. When using Cz-REF as a reference, both the left and right RP followed a straight line at the Cz channel, and it was difficult to discern when movement preparation occurred. However, the RP was clear at the Cz channel when the EEG data was adjusted to a CAR or REST reference. For channels C1 and C2, which are near Cz, we can see that the amplitude of RP was smaller from the Cz-REF reference than from the CAR and REST references. The left RP amplitude was higher on channel C2, while the right RP amplitude was higher on channel C1. On channels FC1, FC2, and FCZ, which are located far from Cz-REF, although a clear RP could be seen in all three references, the RP amplitude of Cz-REF was still smaller than those of CAR and REST. On channels F1, F2, and Fz, which are the farthest from Cz-REF, the RP became unclear compared with channels FC1, FC2, and FCZ, and the RP amplitude also became small in the CAR and REST references. The waveforms of CAR and REST were similar for all nine channels, but the amplitude of the REST reference was slightly higher than that of the CAR reference.

Figures 6, 7 show the same subject's RP voltage topographies for the left and right hand movements in the three references. From these two figures, the following three observations can be made. First, the activation areas of the RP were similar. Second, compared with the other two references, the RP voltage topographies of the Cz-REF reference did not show obvious activation, especially for the primary motor cortex. Finally, the REST reference was as good as the CAR reference for drawing the voltage topographies of the RP. The same results were obtained for the other subjects in the experiment.

Table 1 shows the classification accuracy of the idle and RP periods from 10 participants in three references. High classification accuracy was achieved for all participants except S8, for whom the accuracy was around 70%. The average classification accuracies from the three references were 88.41% (CAR), 87.35% (Cz-REF), and 88.91% (REST), respectively. The REST reference achieved the highest classification accuracy, and the Cz-REF reference achieved the lowest classification accuracy, but the differences among the three references were not statistically significant (p < 0.05).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.