BACKGROUND: An acupuncture treatment can reduce pain sensitivity in patients with chronic whiplash associated disorders (WAD). But it has been hypothesized that many of the experimental results in acupuncture research could be interpreted as stress-induced analgesia. OBJECTIVE: The present study aimed at examining whether acupuncture has an effect on the autonomic nervous system response in patients with chronic WAD and if this response is related to the pain inhibition after an acupuncture session. STUDY DESIGN: Randomized crossover trial with blinded assessor. SETTING: Two private practices. METHODS: Thirty-nine patients with chronic WAD received 2 treatment sessions of identical duration, with acupuncture and relaxation therapy randomly crossed over in the 2 visits. The primary outcome measurement was the registration of autonomic nervous system parameters (heart rate, skin conductance, and heart rate variability parameters) during the administration of experimental pain. Endogenous analgesia was the secondary outcome. RESULTS: Following one acupuncture treatment session, there was a significant change for 2 parameters: the heart rate was slightly reduced and the skin conductance was raised. Comparing the effects of acupuncture and relaxation, no differences were found with respect to the change in any of the autonomic parameters. Further, the reduction in pain sensitivity in response to acupuncture treatment was unrelated to any of the changes in autonomic measurements. LIMITATIONS: The results were observed after only one session of acupuncture. CONCLUSION: In patients with chronic WAD, in response to a single treatment session, no acupuncture specific effects on the autonomic response to pain assessment were present and the analgesia after one session of acupuncture is not caused by stress-induced analgesia but is more likely the result of an acupuncture specific reaction.