BACKGROUND: Many patients with chronic pain, including those with chronic whiplash-associated disorders (WAD), show features of central sensitization. Randomized trials examining whether treatments are able to influence the process of central sensitization in patients with chronic WAD are emerging. Therefore, the present study aimed at examining whether acupuncture results in activation of endogenous analgesia and relief in symptoms in patients with chronic WAD. METHODS: In this randomized crossover pilot trial with blinded assessors, each patient (n = 39) received two treatment sessions of identical duration, with acupuncture and relaxation therapy randomly crossed over in visit 2. Primary outcome measurement included immediate activation of endogenous analgesia i.e., pressure pain sensitivity and conditioned pain modulation. Secondary outcome measurements included pain relief and reduced disability level. RESULTS: Local pressure pain sensitivity at baseline and during conditioned pain modulation decreased significantly more following acupuncture compared with relaxation (time × group interactions: p < 0.001), both in the neck and at a site distinct from the painful region. When comparing the effects of acupuncture versus relaxation, no differences were observed on conditioned pain modulation, temporal summation of pressure pain, neck disability or symptom severity (all p-values >0.05). CONCLUSION: It was shown that one session of acupuncture treatment results in acute improvements in pressure pain sensitivity in the neck and calf of patients with chronic WAD. Acupuncture had no effect on conditioned pain modulation or temporal summation of pressure pain. Both acupuncture and relaxation appear to be well-tolerated treatments for people with chronic WAD. These findings suggest that acupuncture treatment activates endogenous analgesia in patients with chronic WAD.