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Purpose  To investigate the gastrointestinal secretory and enzymatic responses to a liquid meal during in vivo perfusion of the proximal human jejunum. Methods  Human intestinal fluid was collected from the proximal jejunum by single-pass in vivo perfusion (Loc-I-Gut). The fluid was quantitatively collected at 10-min intervals during 90 min while perfusing a nutritional drink at 2 mL/min. Quantification of lipids in the fluid leaving the segment was performed by using novel chromatographic methods. Results  The overall bile acid concentration varied between 0.5 and 8.6 mM with a peak level 40 min after the start of the liquid meal perfusion. The total concentration of phospholipids was between 0.1 and 3.9 mM and there was a rapid degradation of phosphatidylcholine to lysophosphatidylcholine. The tri-, di-, monoglycerides and free fatty acid levels increased sharply in the beginning and reached steady-state levels between 7 and 9.5 mM. Conclusions  There is a rapid secretion of bile in response to food. Most of the dietary lipids are found in the form of their degradation products in vivo in human jejunum. This novel in vivo characterization, based on direct and high-recovery sampling of intestinal fluids, forms a basis for further development of improved in vitro drug dissolution test media.