Acupuncture is known to influence autonomic nervous activity. Acupuncture on Zusanli points has been shown to enhance the regularity of gastric myoelectrical activity and accelerate gastric emptying, partly through the vagal pathway, in dogs. The aim of this study was to evaluate whether atropine-induced autonomic nervous alteration, measured by heart rate variability (HRV), could be amended by electroacupuncture on Zusanli points. HRV measurements were recorded in 15 healthy volunteers before, during and after electroacupuncture. Each subject was studied for three sessions in a randomized sequence, which included electroacupuncture on the Zusanli (St 36) points with or without premedication of atropine and placebo stimulation on a non-acupoint. The analysis of low frequency (LF), high frequency (HF) and LF/HF ratios were compared between different sessions. Serum levels of gastrin, motilin and pancreatic polypeptide (PP) levels were also measured. There was an increase in the LF/HF ratio (indicating increased sympathetic activity) during the post-acupuncture period with 2 Hz of electrical stimulation on the Zusanli acupoints. When IV atropine was used immediately before the electroacupuncture, there was a decrease in the LF power and HF power during the acupuncture and post-acupuncture periods. In addition, there was a significant increase in the LF/HF ratio during the acupuncture and post-acupuncture periods. There was a significant decrease in serum PP in the post-acupuncture period after premedication with IV atropine. In conclusion, atropine-induced HRV change might be mediated via the vagal pathway. However, atropine-induced HRV alteration is not amended by electroacupuncture on Zusanli points.