The milk fat globule membrane (MFGM) is a milk fraction with many potential applications, including addition to infant formulas, where it is suggested to facilitate neural development of the neonate. Severe heat treatment and mechanical processing is known to affect proteins and polar lipids associated with MFGM, with potential detrimental effects on its bioactivity and functionality. The present study describes a more gentle MFGM isolation method based on microfiltration of minimally processed milk, with perspectives for industrial implementation. Efficient separation of fat globules from milk proteins was achieved using polymeric 0.65 µm membranes with a significantly reduced casein and β-lactoglobulin content in the final MFGM isolate, when carried out before heat treatment (80, 85 or 88°C, 15s). Conversely, post-filtration pasteurization resulted in β-lactoglobulin contamination of MFGM isolate and a reduced content of the indigenous MFGM protein PAS 6/7. Heat- and homogenization-induced associations of whey proteins and caseins with the MFGM isolates consisted of both covalent (disulphide) and non-covalent bondings (hydrophobic interactions). Ceramic membranes (1.4 µm, lab scale) out-performed 0.8 µm membranes in terms of flux, fat and protein separation efficiency as well as purity of the MFGM protein composition. At pilot scale, 1.4 µm pore size ceramic membranes resulted in an MFGM isolate containing 7% polar lipids and 30% protein, with a low content of non-MFGM proteins. Applying a mild pasteurization (72°C, 15s) before or after filtration, in this way, did not affect MFGM composition significantly and underlined the importance of choosing pasteurization temperature and time combinations with care to ensure minimal changes to the MFGM isolates. The results point toward an isolation method to obtain less damaged and more pure MFGM material, with the potential of valorising protein-rich permeate streams.