We aimed to evaluate the effect of arousal level in healthy subjects on P50 potential, as the variation in the level of arousal may be a source of variance in the recordings as well as it may provide additional information about the pathology under study. Eleven healthy volunteers participated in the study. A standard auditory P50 potential paradigm was applied. Two stimulation conditions were selected: eyes-open, i.e., high arousal level condition and eyes-closed, i.e., low arousal level condition. P50 component amplitudes in response to both the first (S1) and second stimulus (S2) of the pair, their ratio and difference were evaluated. P50 amplitude in response to S1 was significantly higher during the low arousal closed eyes condition as compared to high arousal open eyes condition. There was no P50 amplitude difference in response to S2 and no arousal effect on gating measures. This prompts for more careful evaluation of patients' arousal level when performing P50 studies and interpreting the results.