Parasympathetic Nervous System (PNS) activity increases while Sympathetic Nervous System (SNS) activity remains relatively stable from wakefulness to NREM sleep. However, it is not clear whether these changes are specifically associated with NREM sleep, or whether they anticipate sleep onset. The latter may occur if ANS activity was influenced by the circadian system. This issue was investigated by conducting spectral analysis of heart beat-to-beat intervals (Periodogram method), collected from 20 healthy male and female subjects at three different times across 24 h; in the morning, just prior to normal sleep onset time, and in slow-wave sleep (SWS). Subjects were supine in all conditions and awake in the first two conditions. The high- and low-frequency peaks, reflecting PNS and SNS activity, respectively, were expressed as proportions of the total power. PNS activity decreased significantly from the morning (0.22) to the presleep period (0.19), before it increased to its maximum during SWS (0.33). In contrast, SNS activity was similar in each of the three conditions (0.07, 0.06, and 0.05 for morning, presleep and SWS, respectively). Thus there do not appear to be changes in PNS activity in anticipation of sleep, as would be predicted on the basis of a circadian influence on the PNS. Instead the increased PNS activity appears to be sleep dependent.