The purpose of the present study was to investigate effect of electro-acupuncture (EA) at different frequencies on autonomic function. Twenty healthy adult volunteers were studied, and underwent 4 sessions of EA (sham, 2Hz, 15Hz, and 50Hz). Sham, 2Hz, 15Hz, and 50Hz EA was applied to the bilateral Leg Three Li (足三里 zú sān lǐ, ST-36) and Upper Great Hollow (上巨虛 shàng jù xū, ST-37) acupoints. The intensity of electrical stimulation was adjusted to obtain visible twitching of the anterior tibial muscle about 2.0-2.5mA except sham without electrical stimulation. The components of heart rate variability (HRV) and blood pressure were measured before EA (BLP), EA (EAP), and post-EA periods (PEP). The results indicated that the natural logarithmic high frequency power (lnHF) of HRV was greater during PEP than during the BLP in the 2Hz EA sessions. The natural logarithmic low frequency power (lnLF) of HRV was greater during the PEP than during the BLP in 15Hz EA sessions, suggesting that 2Hz EA apply to Leg Three Li (足三里 zú sān lǐ, ST-36) and Upper Great Hollow (上巨虛 shàng jù xū, ST-37) acupoints increased vagal activity, whereas 15Hz EA increased sympathetic activity.