INTRODUCTION: Noninvasive positive pressure has been used to treat several diseases. However, the physiological response of the cardiac autonomic system during bilevel positive airway pressure (Bilevel) remains unclear. OBJECTIVE: The aim of this study was to evaluate the heart rate variability (HRV) during Bilevel in young healthy subjects. METHODS: Twenty men underwent 10-minute R-R interval recordings during sham ventilation (SV), Bilevel of 8-15 cmH2O and Bilevel of 13-20 cmH2O. The HRV was analyzed by means of the parallel R-R interval (mean R-Ri), the standard deviation of all R-Ri (SDNN), the root mean square of the squares of the differences between successive R-Ri (rMSSD), the number of successive R-Ri pairs that differ by more than 50 milliseconds (NN50), the percentage of successive R-Ri that differ by more than 50 milliseconds (pNN50), the low frequency (LF), the high frequency (HF) and SD1 and SD2. Additionally, physiological variables, including blood pressure, breathing frequency and end tidal CO2, were collected. Repeated-measures ANOVA and Pearson correlation were used to assess the differences between the three studied conditions and the relationships between the delta of Bilevel at 13-20 cmH2O and sham ventilation of the HRV indexes and the physiological variables, respectively. RESULTS: The R-Ri mean, rMSSD, NN50, pNN50 and SD1 were reduced during Bilevel of 13-20 cmH2O as compared to SV. An R-Ri mean reduction was also observed in Bilevel of 13-20 cmH2O compared to 8-15 cmH2O. Both the R-Ri mean and HF were reduced during Bilevel of 8-15 cmH2O as compared to SV, while the LF increased during application of Bilevel of 8-15 cmH2O as compared to SV. The delta (between Bilevel at 13-20 cmH2O and sham ventilation) of ETCO2 correlated positively with LF, HF, the LF/HF ratio, SDNN, rMSSD and SD1. Acute application of Bilevel was able to alter the cardiac autonomic nervous system, resulting in a reduction in parasympathetic activity and an increase in sympathetic activity and higher level of positive pressure can cause a greater influence on the cardiovascular and respiratory system.