Objectives : With an assumption of the traditional oriental medical theory, we were to evaluate the effect of tonification/sedation-manipulated acupuncture on autonomic nervous activity in healthy subjects. Methods : This study is a randomized cross-over trial. We enrolled healthy subjects, and assigned them randomly into group A or group B. Group A received acupuncture therapy on Shen-Men (He-7) and Nei-Kuan (EH-6) with tonification-manipulation, while Group B received it with sedation-manipulation. Their heart rate variability was monitored for 2 hours. After 3 days of washout period, the subjects were crossed over to the other manipulation method, and the same procedures were performed. Results : There was no statistical significance in the change of RR, SDNN, LF, HF, and LF/HF ratio between the tonification-manipulated and the sedation-manipulated groups. However, HF curve in the tonification manipulated group showed increasing tendency for 15 minutes after acupuncture. Conclusions :We suggest that the traditional tonification-manipulated acupuncture on Shen-Men and Nei-Kuan could increase parasympathetic activity shortly after acupuncture, but further evaluation on a larger sample size is needed to confirm this suggestion.