Patients with acute whiplash-associated disorder (WAD) demonstrate an inefficient endogenous pain inhibition and may experience a dysfunction in autonomic nervous system reactivity to pain. This study compared the autonomic response to painful stimuli between patients with acute and chronic WAD and healthy controls. In addition, the role of the autonomic nervous system for explaining inefficient endogenous pain inhibition was examined in acute WAD. Seventeen patients with acute WAD, 30 patients with chronic WAD, and 31 healthy controls participated in an experiment evaluating the autonomic nervous system at rest and during painful stimuli. Skin conductance and heart rate variability (HRV) parameters were monitored continuously during conditioned pain modulation. A significant autonomic response to pain was present for skin conductance and two HRV parameters in all experimental groups. There was an interaction effect in the skin conductance response to pain but not in HRV responses in any of the groups. In patients with acute WAD, no significant correlations were present between pain, pressure pain thresholds, pain inhibition, and any of the autonomic parameters. This study refutes autonomic dysfunction at rest and in response to pain in acute WAD. The dysfunctional conditioned pain modulation appears unrelated to autonomic responses to pain.