BACKGROUND: Insulin and growth hormone (GH) - 2 vital metabolic regulatory hormones - regulate glucose, lipid, and energy metabolism. These 2 hormones determine substrate and energy metabolism under different living conditions. Shift of day and night affects the clock system and metabolism probably through altered insulin and GH secretion. METHODS: Five-week-old male mice were randomly assigned to a rotating light (RL) group (3-day normal light/dark cycle followed by 4-day reversed light/dark cycle per week) and normal light (NL) group. Body weight and food intake were recorded every week. Series of blood samples were collected for pulsatile GH analysis, glucose tolerance test, and insulin tolerance test at 9, 10, and 11 weeks from the start of intervention, respectively. Indirect calorimetric measurement was performed, and body composition was tested at 12 weeks. Expressions of energy and substrate metabolism-related genes were evaluated in pituitary and liver tissues at the end of 12-week intervention. RESULTS: The RL group had an increased number of GH pulsatile bursts and reduced GH mass/burst. RL also disturbed the GH secretion regularity and mode. It suppressed insulin secretion, which led to a disturbed insulin/GH balance. It was accompanied by the reduced metabolic flexibility and modified gene expression involved in energy balance and substrate metabolism. Indirect calorimeter recording revealed that RL decreased the respiratory exchange ratio (RER) and oxygen consumption at the dark phase, which resulted in an increase in fat mass and free fatty acid levels in circulation. CONCLUSION: RL disturbed pulsatile GH secretion and decreased insulin secretion in male mice with significant impairment in energy, substrate metabolism, and body composition.