Using a newly developed in vivo model measuring glucagon-like peptide-1 (GLP-1) in gut lymphatics in mice, we quantified GLP-1 secretion in vivo after glucose versus fat ingestion with and without concomitant DPP-4 inhibition. The mesenteric lymphatic duct was cannulated in anesthetized C57BL6/J mice and lymph was collected in 30 min intervals. Glucose or fat emulsion (Intralipid®) (0.03, 0.1 or 0.3 kcal) with or without DPP-4-inhibition (NVP DPP728; 10 μmol/kg) was administered by gastric gavage. Basal intact GLP-1 levels were 0.37±0.04 pmol/l (n=61) in lymph compared to 0.07±0.03 in plasma (n=6; P=0.04) and basal DPP-4 activity was 4.7±0.3 pmol/min/μl in lymph (n=23) compared to 22.3±0.9 pmol/min/μl in plasma (n=8; P<0.001). Lymph flow increased from 1.2±0.1 μl/min to 2.3±02μl/min at 30 min after glucose and fat administration, with no difference between type of challenge or dose (n=81). Lymph GLP-1 levels increased calorie-dependently after both glucose and fat but with different time courses in that glucose induced a transient increase which had returned to baseline after 90 min whereas the lipid induced a sustained increase which was still elevated above baseline after 210 min. Lymph GLP-1 appearance during 210 min was two to three-fold higher after glucose (7.4±2.3 fmol at 0.3 kcal) than after isocaloric fat (2.9±0.8 fmol at 0.3 kcal; P<0.001). The slope between caloric load and lymph GLP-1 appearance was, however, identical after glucose and fat. We conclude that lymph GLP-1 is higher than plasma GLP-1 whereas lymph DPP-4 activity is lower than plasma DPP-4 activity and that both glucose and fat clearly stimulate GLP-1 secretion calorie-dependently in vivo but with different time courses.