The objective of this study was to assess the effects of acupuncture applied at Hegu (LI 4) points and Neiguan (PC 6) points on heart rate variability (HRV) in normal subjects under fatigue and non-fatigue states using power spectral analysis. Twenty-nine normal male subjects were randomly divided into three groups. Subjects in Group A and Group B performed a simulated driving task for 3 h and acupuncture needles were then inserted perpendicularly into the LI 4 points in the middle of the dorsal thenar muscle and PC 6 points situated between the tendons of the palmaris longus and carpi radialis muscles for 15 min for Group A but inserted subcutaneously to the acupuncture points for Group B as a control. Acupuncture needles were directly inserted perpendicularly into the LI 4 points and PC 6 points for 15 min for Group C. Stimulations of the acupuncture points induced a significant decrease in heart rate, HRV total power, low frequency (LF) power and ratio of LF to high frequency (HF), and a significant increase in the HF power in normalised units (NU) during the post-stimulation period in the fatigue state. Stimulation of acupuncture points resulted in a significant increase both in the LF and HF powers in absolute units but no significant change in NU was found during the post-stimulation period in the non-fatigue state.