We used spectral analysis of heart rate variability, as a measure of autonomic tone, to determine spectral power differences in infants sleeping supine and prone. We studied 29 infants with a birth weight of 1,915 ± 939 g, at the postconceptional age of 36 ± 2 weeks. We then calculated total power (TP), low-frequency power (LF, 0.03–0.15 Hz), and high-frequency power (HF, 0.5–1.0 Hz). TP corresponds to overall heart rate variability, LF to both sympathetic and parasympathetic activity, and HF to parasympathetic activity only. Median (25th, 75th percentile) TP (beats/min²) in the supine position was 32.60 (23.12, 59.90), which was significantly higher than the prone position of 25.87 (14.94, 35.57). Similarly, LF (beats/min²) in the supine position of 13.82 (8.63, 23.31) was significantly higher than the prone position of 9.79 (5.46, 14.33). No significant difference was seen in the HF. We conclude that the prone position is associated with decreased heart rate variability and probably decreased sympathetic tone, which imply decreased autonomic stability in this position.