Abstract A cross-sectional study was carried out to clarify the physiological features of spontaneous awakening from nocturnal sleep (i.e., whether a child can spontaneously wake up on weekday mornings). The study population comprised 116 children at ages 5 and 6 years. Heart rate variability reflecting cardiac sympathetic and parasympathetic activities was measured. Children's typical bedtimes and wake times for weekdays and the presence/absence of spontaneous awakening from nocturnal sleep were reported by parents, and information about obligatory naptimes was provided by preschool teachers. The mean total sleep duration in the children was 625 ± 56 (standard deviation) min. Total and nocturnal sleep durations were significantly shorter in 52 children without spontaneous awakening than in 64 children with it. Similarly, the parasympathetic activity was significantly lower in the children without spontaneous awakening, even in using analysis of covariance. Heart rate was significantly increased in the children without spontaneous awakening, but neither total nor nocturnal sleep durations were significant covariates in the analysis of covariance. In conclusion, the absence of spontaneous awakening from nocturnal sleep in preschool children is suggested to be characterized by short sleep duration, parasympathetic hypoactivity, and elevated heart rate.