To examine whether habituation confounds the study of whiplash injury using human subjects, we quantified changes in the magnitude and temporal development of the neck muscle electromyogram and peak linear and angular head/torso kinematics of subjects exposed to sequential whiplash-like perturbations. Forty-four seated subjects (23F, 21M) underwent 11 consecutive forward horizontal perturbations (peak sled acceleration=1.5 g). Electromyographic (EMG) activity was recorded over the sternocleidomastoid (SCM) and cervical paraspinal (PARA) muscles with surface electrodes, and head and torso kinematics were measured using linear and angular accelerometers and a 3D motion analysis system. EMG onset occurred at reflex latencies (67–75 ms in SCM) and did not vary with repeated perturbations. EMG amplitude was significantly attenuated by the second perturbation in PARA muscles and by the third perturbation in SCM muscles. The mean decrement in EMG amplitude between the first trial and the mean of the last five trials was between 41% and 64%. Related kinematic changes ranged from a 21% increase in head extension angle to a 29% decrease in forward acceleration at the forehead, and were also significantly different by the second exposure in some variables. Although a wider range of perturbation intensities and inter-perturbation intervals need to be studied, the significant changes observed in both muscle and kinematic variables by the second perturbation indicated that habituation was a potential confounder of whiplash injury studies using repeated perturbations of human subjects.