Protein ingestion after injection of the GLP-1 receptor agonist Exendin-4 (Ex-4) causes hyperglycemia in rats. The objectives of this study were to determine the components of protein digestion responsible for this effect and to associate it with changes in the concentrations of other metabolites and hormones. Two experiments were conducted. In the first experiment, food-deprived rats were gavaged with intact whey (WP) or albumin protein, their hydrolysates, or amino acid mixtures (1 g/2.5 ml), or water 5 min after injection of either phosphate buffer saline (PBS) or Ex-4 (0.5 µg/rat). Tail vein blood was analyzed for glucose over two hours. In the second experiment, food-deprived rats were gavaged with WP with or without Ex-4. Groups of conscious rats were sacrificed by decapitation either before, or at selected times after gavage. Plasma concentrations of glucose, amino acids, free fatty acids (FFA), glycerol, insulin, glucagon and leptin were measured. In experiment 1, blood glucose was higher when intact proteins and protein hydrolysates, but not amino acid mixtures, were given with than without Ex-4 (p<0.05). In experiment 2, concentrations of glucose, FFA, and the ratio of tyrosine/branched-chain amino acid were higher (p<0.01), but leptin and essential amino acid concentrations were lower (p<0.05), and insulin, glucagon and glycerol were similar when WP was given with than without Ex-4. We conclude that the hyperglycemia caused by the administration of Ex-4 concurrently with dietary protein arises from the action of peptides released during digestion, and their interaction with Ex-4 in the regulation of glucose, fatty acid, and amino acid metabolism.