Effects of high-pressure homogenisation (HPH, 100 MPa) at different temperatures (4–60 °C) or conventional homogenisation (20 MPa, 60 °C) on size of milk fat globules (D32) and content of protein and phospholipids in milk fat globule membrane (MFGM) in standardised milk, buttermilk and a mixture of milk and buttermilk were compared. HPH decreased D32 and promoted the adsorption of casein at ≥ 4 °C, β-lactoglobulin in milk and buttermilk at ≥ 40 °C and ≥20 °C, respectively, and α-lactalbumin at 60 °C, on the surface of milk fat globules. The content of milk proteins adsorbed on MFGM increased with increasing temperature and pressure in milk, and, to a lesser extent, in buttermilk. The content of phospholipids in buttermilk was 2-fold higher than in milk. HPH influenced the content of sphingomyelin (increased) and phosphatidylcholine (decreased) in MFGM in milk. Conventional homogenisation increased the phosphatidylethanolamine content in MFGM in buttermilk.