Paced breathing (PB) around 0.25 Hz has been advocated as a means to avoid confounding and standardize measurements in short-term investigations of autonomic cardiovascular regulation. Controversy remains, however, as to whether it causes any alteration in autonomic control. We addressed this issue in 40 supine middle-aged healthy volunteers by assessing the changes induced by PB (0.25 Hz for 8 min) on i) ventilatory parameters, ii) the indexes of autonomic control of cardiovascular function and iii) the spectral indexes of cardiovascular variability. Subjects were grouped into group 1 (N=31) if spontaneous breathing was regular and within the high-frequency (HF) band (0.15 - 0.45 Hz), or group 2 (N=9) if it was irregular or slow (<0.15 Hz). In both groups PB was accompanied by an increase in minute ventilation (both p< 0.01), while tidal volume increased only in group 1 (p=0.0003). End-tidal CO2 decreased by (median (lower quartile, upper quartile)) -0.2 (-0.5 , -0.1)% (group 1, p<0.0001) and -0.6 (-0.8 , -0.5)% (group 2, p=0.008). Mean RR interval, systolic and diastolic pressure remained remarkably stable (all p 0.13, both groups). No significant changes were observed in spectral indexes of RR and pressure variability (all p 0.12, measured only in group 1 to avoid confounding), except in the HF power of pressure signals, which significantly increased (all p<0.05) in association with increased tidal volume. In conclusion, PB at 0.25 Hz causes a slight hyperventilation and does not affect traditional indexes of autonomic control nor, in subjects with spontaneous breathing in the HF band, most relevant spectral indexes of cardiovascular variability. These findings support the notion that PB does not alter cardiovascular autonomic regulation compared to spontaneous breathing.