To examine the effect on serum lipase activity and protein concentration of intravenous infusions of glucagon-like peptide-1 (GLP-1) and peptide YY (PYY3-36) and of an ad libitum meal in healthy overweight men. Twenty-five healthy, male subjects participated in this randomized, double-blinded, placebo-controlled 4-arm crossover study (Body Mass Index (BMI): 29 ± 3 kg/m(2), age: 33 ± 9 years). On separate days, the subjects received a 150-min intravenous infusion of either (1) 0.8 pmol/kg/min PYY3-36, (2) 1.0 pmol/kg/min GLP-1, (3) 1 + 2, or (4) placebo. Samples were collected throughout the infusion and after intake of an ad libitum meal for measurement of serum lipase. Serum lipase levels measured by enzyme-linked immunosorbent assay (ELISA) following mono-infusions of GLP-1 and PYY3-36 were comparable to serum lipase levels following placebo (P = 0.054 and P = 0.873, respectively). Following the co-infusion of GLP-1 and PYY3-36, serum lipase levels measured by ELISA decreased over time compared to placebo (P = 0.012). However, the between-group difference was not consistent when each time point was analyzed separately. On the placebo day, serum lipase levels measured by ELISA after an ad libitum meal rose slightly compared to the preprandial values (P = 0.003). There was strong correlation between serum lipase levels measured by ELISA and LIPC Lipase colorimetric assay (COBAS) (0.94 < r; <0.0001). Infusions of GLP-1 and PYY3-36, separately or in combination, did not increase serum lipase. However, a small increase in serum lipase may occur in response to a meal.