Study Objective: To determine the hormonal effects of reducing sleep duration under controlled feeding conditions. Design: Randomized, crossover study. Setting: Inpatient. Participants: Twenty-seven normal weight, 30- to 45-yr-old men and women habitually sleeping 7-9 hr/night. Intervention: Participants were studied under two sleep conditions: short (4 hr in bed) or habitual (9 hr in bed) sleep. A controlled diet was provided for each 4-day study period. Measurements and Results: Fasting blood samples were obtained daily and frequent blood samples were obtained throughout day 4. The main outcomes measures included glucose, insulin, leptin, ghrelin, adiponectin, total glucagon-like peptide 1 (GLP-1) and peptide YY3-36 (PYY3-36) concentrations. Body weights were reduced by 2.2 ± 0.4 lb and 1.7 ± 0.4 lb during the habitual and short sleep phases, respectively (both P < 0.0001). There was no effect of sleep duration on glucose, insulin, and leptin profiles (all P > 0.05). Ghrelin and GLP-1 responses differed by sex. Short sleep increased fasting (P = 0.054) and morning (08:00-12:00) (P = 0.042) total ghrelin in men but not women. The reverse was observed for GLP-1: afternoon levels (12:30-19:00) were lower (P = 0.016) after short sleep compared with habitual sleep in women but not men. Conclusions: These data suggest that, in the context of negative energy balance, short sleep does not lead to a state of increased insulin resistance, but may predispose to overeating via separate mechanisms in men and women.