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The present study aimed to evaluate, in gilthead sea bream, the effect of dietary protein:energy (P/E) ratio and non‐protein energy (NPE) source on the post‐prandial profile of plasmatic parameters, hepatic energy reserves, enzymatic activities of digestive enzymes and hepatic key enzymes activities of the glycolytic (HK and GK), lipogenic (G6PDH and FAS), gluconeogenic (FBPase) and amino acid (GDH, AST and ALT) metabolic pathways. Fish (44 ± 0.05 g) were fed for 34 days four diets with high (HP) or low (LP) P/E ratios and lipid (L) or starch (S) as NPE source (diets HPL, HPS, LPL and LPS, respectively). No differences in growth performance were observed between groups. However, FI was lower, and FE was higher in fish fed the HPS diet than in other groups. Plasma triglycerides and glycaemia were affected by diet composition. Hepatic glycogen and lipid content of fish fed the HPL diet were lower than other groups at different post‐prandial times. Digestive enzymes peaked between 6 and 12 h after feeding. HK and GK activities peaked at 6 h after feeding and followed the amylase activity peak. Glycolysis and lipogenesis enzyme activities were higher in fish fed the starch than lipid diets. Gluconeogenesis enzyme activity was high, and amino acid catabolism enzyme activity was lower in fish fed the LP than HP diets. In conclusion, dietary NPE sources and P/E ratio affected the metabolic responses in gilthead sea bream; overall, NPE sources affected carbohydrate metabolism and lipogenesis, whereas P/E ratio mainly affected gluconeogenesis and amino acid catabolism. [ABSTRACT FROM AUTHOR]