There is a lack of knowledge available on how cats adjust their macronutrient partitioning due to the consumption of single-macronutrient meals. The objective of this study was to evaluate consumption of a single meal of ingredients that contained foods of strictly carbohydrates (CHO), fat (FAT) or protein (PRO), on energy expenditure (EE) and macronutrient metabolism in cats. Ten domestic shorthair adult cats (1·9 years; 4·12 kg) were fed 22–24 g of chicken fat (FAT), 56–62 g of whey protein solution (PRO) or 54–56 g of cornstarch solution (CHO) for a single day in a randomised complete block design. Indirect calorimetry was conducted for 24 h post-feeding. Mean average EE over 24 h was highest in cats fed PRO (44 kcal/kg BW) and FAT (43 kcal/kg BW) compared with that in cats fed CHO (42 kcal/kg BW; P < 0·01). During 0 to 4 h, cats fed FAT had greater EE (49 kcal/kg BW), suggesting that cats respond to oxidising more dietary fat over protein in the early postprandial stage. Mean 24 h respiratory quotient (RQ) was greatest for cats fed CHO (0·76) followed by PRO (0·75) and FAT (0·74; P < 0·05). During 4 to 8 h, the RQ of cats fed PRO was the greatest (0·77), suggesting that cats initially increase gluconeogenesis from amino acids for subsequent glucose oxidation. In comparison to omnivores and herbivores, obligate carnivores have unique responses to single macronutrient intake, where they apparently generate energy from carbohydrate metabolism and rely more on gluconeogenic precursors.