Hatha yoga is commonly practiced in Western countries and is claimed to reduce risk of cardiovascular disease. The purpose of this study was to evaluate and compare time-domain and frequency-domain metrics of heart rate variability (HRV) in Hatha yoga practitioners and healthy controls. This cross-sectional study, which was conducted at a regional university and community wellness center, included convenience sampling of 19 Hatha yoga practitioners and 8 healthy controls. Using a lead II ECG system, 10 minutes of electrocardiogram (ECG) recording was collected for each participant. Artifact-free, 5-minute signals were used to derive time-domain and frequency-domain measures of HRV. The mean duration of Hatha yoga practice among practitioners was 11.47 ± 8 years. Demographic and anthropometric characteristics did not differ significantly between groups. Compared with the control group, the yoga group had significantly greater mean high frequency (HF) power (859.2 ± 1342.1 vs 175.5 ± 121.1; P = .04) and mean HF normalized units (nu) (57.0 ± 16.6 vs 36.7 ± 13.4; P = .02) and a significantly lower low frequency (LF)/HF ratio (1.1 ± 0.5 vs 2.2 ± 1.1; P = .01). No significant intergroup differences were observed for LF power, LF nu, or any time-domain measures of HRV. These results lack generalizability due to small sample size and lack of blinded assessment of outcome measures. Hatha yoga practitioners showed parasympathetic predominance compared with healthy controls. Analyzing frequency-domain HRV metrics enables detecting changes in cardiac autonomic function earlier than by analysis of time-domain metrics. Parasympathetic predominance demonstrated in the yoga group suggests Hatha yoga practitioners may be at lower risk for stress-related comorbidities.