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Background: Broiler chickens are compulsive feeders that become obese as juveniles and are thus a unique model for metabolic disorders in humans. However, little is known about the relationship between dietary composition, fasting and refeeding and adipose tissue physiology in chicks. Our objective was to determine how dietary macronutrient composition and fasting and refeeding affect chick adipose physiology during the early post-hatch period. Methods: Chicks were fed one of three isocaloric diets after hatch: high-carbohydrate (HC; control), high-fat (HF; 30% of ME from soybean oil) or high-protein (HP; 25% vs. 22% crude protein). At 4 days post-hatch, chicks were fed (continuous ad libitum access to food), fasted (3 h food withdrawal), or refed (fasted for 3 h and refed for 1 h). Subcutaneous, clavicular, and abdominal adipose tissue was collected for histological analysis and to measure gene expression, and plasma to measure non-esterified fatty acid (NEFA) concentrations (n = 6–10 per group). Results: Adipose tissue weights were reduced in chicks that were fed the HP diet and adipocyte diameter was greater in the adipose tissue of chicks that ate the HF diet. Consumption of diets differing in protein and fat content also affected gene expression; mRNAs encoding fatty acid binding protein 4 and a lipolytic enzyme, monoglyceride lipase, were greater in chicks fed the HC and HF than HP diet in all three adipose tissue depots. Fasting influenced gene expression in a depot-dependent manner, where most fasting and refeeding-induced changes were observed in the clavicular fat of chicks that consumed the HC diet. Fasting increased plasma NEFA concentrations in chicks fed the HC and HP diets. Conclusions: The decreased adipose tissue deposition in chicks fed the HP diet is likely explained by decreased rates of adipogenesis. Consumption of the HF diet was associated with greater adipose tissue deposition and larger adipocytes, likely as a result of greater rates of adipocyte hypertrophy. The depot-dependent effects of diet and fasting on gene expression may help explain mechanisms underlying metabolic distinctions among subcutaneous and visceral fat depots in humans. Published version