OBJECTIVES: To study the effect of acupuncture on heart rate variability (HRV) by using linear and non-linear methods of analysis. METHODS: 40 patients were recruited consecutively, including patients with insomnia, stomachache, diarrhea, dizziness, cervical syndrome, lower back pain, gonarthritis, peripheral facial paralysis, post-traumatic organic brain syndrome and urinary retention. Different acupoint prescriptions were used, according to the textbook for 5-years\' education on traditional Chinese medicine specialty, which is used in Chinese Universities. HRV was recorded before, during, and after acupuncture. RESULTS: Acupuncture substantially reduced variability, causing a 41% reduction in the standard deviation. Using a Fourier analysis, the variances both in the low frequency (LF) and the high frequency (HF) ranges were markedly reduced, but the LF/HF ratio (an indication of sympatho-vagal balance) was not altered. The HR was unchanged. The sample entropy, which is a measure of the complexity of time series, was significantly increased (+35%). CONCLUSIONS: Acupuncture produced a pattern of changes different from that seen in pathological conditions, where increased variability and reduced complexity is expected.