A sequential in vitro model of digestion was used to investigate the changes in the physicochemical properties of emulsions during gastrointestinal transit. Oil-in-water emulsions were prepared with whey protein isolate (WPI) or soy protein isolate (SPI) at the same protein concentration (1.5%). Despite pepsinolysis of both proteins during the gastric phase, emulsions stabilized with WPI were more stable compared to those prepared with SPI. For both emulsions, the size of the oil droplets, which plays a critical role in lipid digestion, was extensively altered during the duodenal phase due to the presence of bile salts (BS) and phospholipids (PL). As shown by ζ-potential measurements, the results suggested the displacement of both proteins from the interface by BS; however, the displacement was much faster for the WPI-emulsions. The change in interfacial composition of the oil droplets was significantly affected by inclusion of PL and phospholipase A2 (PLA2) in the in vitro digestion model. The interfacial activity of pancreatic triglyceride lipase (PTL) was markedly affected in the presence of the surface-active compounds present in the digestive fluids, including BS, PL, colipase (COL) and PLA2. A higher percentage of lipid hydrolysis was obtained in the presence of COL and PLA2 than with BS alone or mixed BS–PL. SPI-emulsions consistently showed a higher degree of lipolysis compared to the WPI-emulsions regardless of the in vitro digestion model used. The results support the conclusion that the interfacial composition of the original emulsion plays a major role in determining the extent of lipolysis.