This study examined the effects of craniosacral therapy (CST) on heart rate variability (HRV) in 31 patients with subjective discomforts. HRV was measured before and after a 30-minute control rest period and a 30-minute CST session on consecutive days using a mobile device. Standard deviation of heart rate intervals (SDNN) and total power (TP), indicators of autonomic nervous system activity, increased significantly after CST but not the control rest. Heart rate also decreased significantly after CST compared to rest. However, interactions between treatment and HRV changes were not fully statistically significant. The study provides preliminary evidence that CST may positively influence autonomic nervous system regulation.