Background: Spontaneous baroreflex method allows continuous assessment of cardiovagal reflex function within resting blood pressure, but effects of nitrous oxide, per se, on the spontaneous baroreflex response remain unknown. This study was designed to determine the effects of nitrous oxide on spontaneous baroreflex gain and heart rate variability assessed by power spectral analysis in humans. Methods: Electrocardiogram and non-invasive blood pressure were monitored in 12 healthy volunteers before and during a 15-min inhalation of 67% nitrous oxide in oxygen, while spontaneous respiration was maintained. Least-square regression analysis relating R-R interval and systolic blood pressure was performed to obtain spontaneous baroreflex gains. Heart rate variability was analyzed using fast Fourier transformation. Results: Nitrous oxide did not significantly alter spontaneous baroreflex gains, which correlated well with high-frequency power (0.15–0.4 Hz) of heart rate variability before and during nitrous oxide inhalation. Conclusion: Our results indicate that (a) cardiovagal reflex response is not affected by nitrous oxide, per se, and (b) spontaneous baroreflex responses closely reflect beat-to-beat dynamic modulation of the cardiac cycle by the parasympathetic nervous system during inhalation of 67% nitrous oxide.