BACKGROUND: Renal nerve stimulation (RNS) is used to localize sympathetic nerve tissue for selective renal nerve sympathetic denervation (RDN). Examination of heart rate variability (HRV) provides a way to assess the state of the autonomic nervous system. The current study aimed to examine the acute changes in HRV caused by RNS before and after RDN. METHODS AND RESULTS: 30 patients with hypertension referred for RDN were included. RNS was performed under general anesthesia before and after RDN. Heart rate (HR) and blood pressure (BP) were continuously monitored. HRV characteristics were assessed 1 min before and after RNS and RDN. RNS before RDN elicited a maximum increase in systolic BP of 45 (+/- 22) mmHg which was attenuated to 13 (+/- 12) mmHg (p < 0.001) after RDN. RNS before RDN decreased the sinus cycle length from 1210 (+/- 201) ms to 1170 (+/- 203) ms (p = 0.03), after RDN this effect was blunted (p = 0.59). The LF/HF ratio in response to RNS changed from 鈭?+ 0.448 (+/- 0.550) before RDN to 鈭?- 0.656 (+/- 0.252) after RDN (p = 0.02). Selecting patients off beta-blockade (n = 11), the RNS-induced changes in HRV components before versus after RDN were more pronounced (LF/HF ratio 鈭?+ 0.900 +/- 1.171 versus 鈭?- 0.828 +/- 0.519, p = 0.01), whereas changes in HRV parameters in patients on beta-blockade (n = 19) were no longer significant. In patients with diabetes mellitus (n = 7), RNS induced no changes in HRV parameters (LF/HF ratio 鈭?- 0.039 +/- 0.103 versus 鈭?- 0.460 +/- 0.491, p = 0.92). CONCLUSION: RNS induces changes in HRV suggesting increased sympathetic activity. Conversely, after RDN, the RNS-induced changes in HRV suggesting a lower sympathetic autonomic balance. These changes were most pronounced in beta-blocker naive patients and not present in patients with diabetes mellitus. These findings could support RNS-guided RDN to optimize results.