BACKGROUND This study examined how osteopathic techniques, including compression of the fourth ventricle (CV4) and rib raising (RR), influence autonomic nervous system (ANS) activity using heart rate variability (HRV) as a measurement tool. This study aimed to evaluate the effects of the fourth ventricle compression technique and the rib raising osteopathic technique on the activity of the autonomic nervous system measured by heart rate variability in 100 healthy adults. MATERIAL AND METHODS A total of 100 healthy participants were divided into 3 groups: CV4+RR (33 subjects), CV4 only (34 subjects), and a control group (33 subjects) undergoing a simulated procedure. Three 30-minute sessions were performed. HRV was recorded during a 5-minute seated session to assess ANS activity. RESULTS While no significant differences in the overall HRV parameter were observed between groups, significant changes were found in specific frequency domain parameters. The low-frequency (LF) parameter differed significantly between groups (p=0.005). A significant interaction effect was observed for the high-frequency (HF) parameter (F2=(4,194)=3.262; p=0.013), indicating group-dependent variations. Additionally, the LF/HF ratio showed a significant interaction effect between group membership and measurement time points (F2=(4,194)=5.39; p<0.001), highlighting differences across groups and time points. CONCLUSIONS The findings demonstrate that osteopathic techniques, particularly the combined CV4+RR approach, significantly affect certain HRV parameters, such as LF%, HF%, and the LF/HF ratio. The CV4+RR group exhibited notable changes compared to the control group, which showed no significant changes. These results suggest the potential effectiveness of the applied interventions in modulating ANS activity.