The effect of therapeutic ultrasound (US) on nervous system is controversial and the effect on autonomic nervous system is not clear. Therefore, the present placebo-controlled trial was planned to investigate the effects of therapeutic US application on right-side stellate ganglion, by using analysis of heart rate variability (HRV). A total 12 healthy volunteers were included in the study. RR intervals were recorded for 5 min before and after the US application, in supine and sitting positions. All procedures were repeated in all participants with sham US one week later. The heart rate (HR) was obtained by time-domain analysis and low frequency (LF) power (%), high frequency (HF) power (%) and LF/HF ratio values were obtained by frequency-domain (power spectral density) analysis. After the US application, there was a decrease in the HR (p = 0.002) and the HF power (%) component (p = 0.015) in supine position and a decrease in HR (p = 0.002) and LF/HF ratio (p = 0.028) in sitting position. There was no significant difference after the sham US application. In conclusion, we observed that therapeutic US application on stellate ganglion causes alterations on HRV parameters.