Objective: During acupuncture stimulation, autonomic nervous system (ANS) function changes toward being parasympathetic-dominant, with a transient decrease in heart rate (HR). The aim of this research was to determine the relationships between cortical relaxation and vigilance as observed on background electroencephalograms (EEGs), HR, and ANS function during deep acupuncture. Materials and Methods: This comparative study was conducted at Teikyo Heisei University, in Toshima-ku, Tokyo, Japan, with 27 healthy male volunteers. After resting for 20 minutes, the men received manual acupuncture at LI 10 on the left forearm for 2 minutes at a depth of 15-20 mm at a 1-Hz frequency while undergoing concurrent EEG and electrocardiography (ECG) monitoring. Each subject described his level of comfort during acupuncture. HR; power ratios (normalized units [n.u.]) of EEG alpha, beta, delta, and theta waves; and HR variability (HRV) indices were calculated. Results: In the subgroup who experienced discomfort, delta and theta n.u. were decreased while alpha n.u. were increased, indicating increased vigilance and decreased relaxation. In the subgroup who experienced comfort, there were no significant changes. HRV indices suggested parasympathetic-dominant changes in both subgroups. Weak correlations were observed between a decrease of theta n.u. and sympathetic-dominant changes in HRV indices. Conclusions: Alterations in background EEG activities were not the primary factors changing ANS function to parasympathetic-dominant and decreasing HR, but these alterations related to a weak secondary factor changing ANS function. EEG activity by which cortical relaxation and vigilance were represented was the weak secondary factor changing ANS function during acupuncture; the primary factor might be supraspinal reflection.