Values reported for basal hepatic glucose production and glucose utilization do not reflect metabolic changes occurring during sleep. To determine the effect of sleep with its associated lowered metabolic rate and thermogenesis on glucose kinetics and gluconeogenic substrate availability, 11 normal volunteers underwent an overnight study in which [3-3H]glucose was infused. Despite decreased insulin secretion, a fall in hepatic glucose output was observed with sleep that was synchronous with a reduction in glucose utilization and lipolysis (decreased plasma glycerol and free fatty acids). When activity was increased, these parameters rose toward previously reported basal levels. Prevention of sleep in 6 additional subjects attenuated the fall in glucose utilization and production as well as the fall in glycerol and free fatty acids despite similar insulin and counterregulatory hormone profiles. We suggest that sleep-associated metabolic changes produce a peripheral signal(s) that modulates hepatic glucose production in humans.