BACKGROUND: The relative contributions of fat and protein to the incretin effect are still largely unknown. OBJECTIVE: This study assessed the incretin effects elicited by a mixed meal, and by its fat and protein components alone, with the use of a hyperglycemic clamp combined with oral nutrients. DESIGN: Eight healthy volunteers were studied over 6 h after ingestion of a sandwich containing 1) dried meat, butter, and white bread; 2) dried meat alone; 3) butter alone; or 4) no meal (fasting control). Meals were ingested during a hyperglycemic clamp, and the incretin effect was calculated as the increment in plasma insulin after food intake relative to the concentrations observed during the control study. RESULTS: A significant augmentation of postprandial insulin secretion, independent of plasma glycemia, occurred after ingestion of the mixed nutrients and the lipid component of the mixed meal (203 ± 20.7% and 167.4 ± 22.9% of control, respectively; both P < 0.05), whereas the protein component did not induce a significant incretin effect (129.0 ± 7.9% of control; P = 0.6) CONCLUSIONS: Fat ingestion, in an amount typical of a standard meal, increases insulin secretion during physiologic hyperglycemia and thus contributes to the incretin effect. In contrast, ingestion of protein typical of normal meals does not contribute to the augmentation of postprandial insulin secretion. This trial was registered at clinicaltrials.gov as NCT00869453.