Objectives: Osteopathic manipulative treatment (OMT) focused on the upper cervical spine is theorized to affect the function of the vagus nerve and thereby influence the parasympathetic branch of the autonomic nervous system. This study was designed to determine the acute effect of upper cervical spine manipulation on cardiac autonomic control as measured by heart rate variability. Design: Nineteen healthy, young adult subjects underwent three different experimental interventions administered in random order: cervical OMT, sham manipulation, and time control. Six minutes of electrocardiographic data were collected before and after each intervention, and heart rate variability was assessed by both time-domain and frequency-domain measures. Results: No differences in resting heart rate or any measure of heart rate variability were observed between the baseline periods prior to each intervention. The OMT protocol resulted in an increase in the standard deviation of the normal-to-normal intervals (0.12±0.082 seconds, p<0.01), an increase in the high frequency spectral power (p=0.03), and a decrease in the low/high frequency spectral ratio (p=0.01) relative to the sham and time control conditions. No significant differences between sham and time control were observed (p>0.11 for all variables). Conclusions: These data support the hypothesis that upper cervical spine manipulation can acutely affect measures of heart rate variability in healthy individuals.