Background: Many studies have been conducted to assess the effects of fentanyl on the autonomic nervous system (ANS) by heart rate variability (HRV) analysis, but disagreement on the results is still present due to confounding influences introduced by concomitant administration of other drugs or opioid-related transitory respiratory depression. We performed a single-drug controlled-breathing study on healthy volunteers to assess the impact of fentanyl on ANS. Methods: HRV as a measure of sympathovagal balance was prospectively analyzed with an autoregressive model in 11 subjects during spontaneous and paced breathing (PB) at 20 breaths/min both before and after fentanyl 1 mcg/kg administration. HRV total power, sympathovagal balance (low-frequency/high-frequency ratio) and normalized spectral powers were considered (LFnu, HFnu). Results: Fentanyl led to a reduction of LFnu (from 55.2±23.3 to 43.2±24.1, P<0.05) without HFnu increase during PB. A decrease in R–R interval variance (from 3345.6±3333.4 to 1806.9±1328.6 ms2, P<0.05) was shown after fentanyl administration during spontaneous but not PB. PB alone decreased the HRV total power and R–R interval variance. Conclusions: Low-dose fentanyl administration in healthy volunteers leads to sympathetic and overall ANS modulation decrease, with a trend toward vagal activation.