Background: Autonomic rehabilitation using osteopathic manipulative treatment (OMT) to stimulate the vagus nerve may be a good adjunct therapy by physiatrists who treat dysautonomia. Heart rate variability (HRV) may provide quantitative evidence for the benefits of OMT on autonomic nervous system function. Elevated HRV indicates optimal health and reduced mortality risk, whereas low HRV is associated with stress, pain, and chronic disease pathology. Objective: To analyze the impact of modified occipitomastoid suture v-spread OMT technique on vagal tone by measuring HRV. Design: Within-participant design with 5-minute HRV measurement pre- and post-OMT treatment. Setting: Clinical examination suites. Participants: Thirty healthy osteopathic medical students. Interventions: A modified occipitomastoid suture v-spread treating somatic dysfunction by osteopathic physicians trained in osteopathic neuromusculoskeletal medicine. Main outcome measures: HRV variables related to vagal tone including root mean square of successive differences (RMSSD), percentage of successive normal sinus RR intervals >50 ms (pNN50), high frequency (HF), Parasympathetic Nervous System Index (PNSI), and Sympathetic Nervous System Index (SNSI). Repeated measures t-test analyzed the difference in mean HRV values after OMT. Results: There were statistically significant increases in each of the HRV measures after OMT. Participants had a mean (95% confidence interval) RMSSD of 50.5 ms (38.3-62.8) at baseline and 55.0 ms (41.2-68.7, p = .013) post-OMT. pNN50 was 24.5% (17.6-31.3, n = 30) at baseline and 28.2% (20.8-35.6, p = .003) post-OMT. HF was 1549.6 ms2 (389.8-2709.4) at baseline and 1901.8 ms2 (618.2-3185.4, p = .103) post-OMT. PNSI was -0.1 (-0.5 to 0.2) at baseline and 0.1 (-0.3 to 1.5, p < .01) post-OMT, and SNSI was 0.2 (-0.1 to 0.6) at baseline and 0.05 (-0.3 to 0.4, p < .01) post-OMT. Normalization, due to high baseline variance, results include RMSSD 1.1 ms (1.0 to 1.1, p = .012), pNN50 1.3 (1.1 to 1.4, p = .011), and HF 1.2 ms2 (1.1 to 1.4, p = .018).