The objective was to evaluate the effects of dietary macronutrients and feeding frequency on blood glucose, insulin, total ghrelin and leptin. A total of twelve adult lean neutered male cats were used in three tests, all cross-over studies composed of a 15 d adaptation and blood sampling on day 16. In trial 1, differences between two- and four-meal feeding were tested. On day 16, blood samples were collected every 2 h for 24 h. In trial 2, macronutrient boluses were tested. Instead of the control diet, the morning meal on day 16 was replaced with an isoenergetic bolus of carbohydrate (maltodextrin), protein (chicken meat), fat or water. Fasted and ten postprandial blood samples were collected. In trial 3, diets high in fat (HF), protein (HP), carbohydrate (HC) or a control diet were tested. On day 16, fasted and ten postprandial blood samples were collected. Data were analysed to identify baseline and AUC changes. Cats fed four meals daily had greater (P = 0·03) leptin incremental AUC0–24 h compared with cats fed twice daily. The carbohydrate bolus increased glucose (P < 0·001) and insulin (P < 0·001) incremental AUC0–6 h and tended to increase (P = 0·09) leptin net AUC0–6 h. Cats fed the control and HC diets had greater (P = 0·03) glucose incremental AUC compared with the HF and HP conditions. Circulating hormone data were highly variable and indicated changes due to dietary macronutrients and feeding frequency, but further study is needed to identify impacts on appetite and contributing mechanisms.