Background: Night work is becoming more common and shift workers display several metabolic disturbances. Aim To study the endocrine responses in relation to time of day during a 24-h period and how dietary macronutrient composition affects these responses. Design: Seven males (26–43 y and 19.9–26.6 kg · m⁻²) were studied in a crossover design. Isocaloric diets described as high-carbohydrates (HC; 65 energy percent (E%) carbohydrates and 20E% fat) or high-fat (HF; 40E% carbohydrates and 45E% fat) were given. After a 6-day diet adjustment period, the subjects were kept awake for 24 h in a metabolic unit and were served an isocaloric meal (continuation of respective diet) every 4-h. Blood samples were taken throughout the 24-h period. Results: Insulin and leptin responses to meal intake differed with respect to time of day (p < 0.05). Time of day affected glucagon, thyroid stimulating hormone (TSH), free thyroxin (fT4), total triiodothyronine (tT3), cortisol, chromogranin A (CgA) and pancreatic polypeptide (PP) concentrations (p < 0.05). Meal intake decreased cortisol concentration after meals at 0800, 1200 and 0400 but not at 1600, 2000 and 0000 h. The PP's postprandial increase was greater during 0800–1600 h compared to 2000–0800 h. With the HC meals, lower glucagon and CgA concentrations (p < 0.05), and a tendency for lower tT3 concentrations (p = 0.053) were observed compared to the HF meals. Conclusion: Insulin, PP, TSH, fT4, cortisol and leptin responses to meal intake differed with respect to time of day. The decreased evening/nocturnal responses of cortisol and PP to meal intake indicate that nocturnal eating and night work might have health implications.