Pepsin diffusion in food particles during gastric digestion is one of the main factors limiting proteolysis kinetics. Diffusion coefficients of pepsin are needed as input parameters for in silico models of digestion, but no values are currently available in real foods. The challenge of this study was to apply the Fluorescent Recovery After Photobleaching (FRAP) technique to determine diffusion coefficients of fluorescently labeled (FITC)-pepsin in four realistic food matrices with complex and heterogeneous structures (Custard, Pudding, Sponge cake and Biscuit), but an identical composition on a dry matter basis. The effective diffusion coefficients determined for FITCpepsin at 37 • C ranged from 48 ± 14 to 2 ± 1 μm 2. s-1 for Custard and Biscuit, respectively. A modelling approach based on the stretched exponential equation generated a very good fit of the experimental dataset as a function of dry matter content of the matrix.