MFGM as a complex mixture of proteins and phospholipids, abundant in the buttermilk and can be used in pharmaceutical and functional food applications. The proteins in the MFGM have been shown to have bioactive properties that are beneficial for human health. Two of these proteins, lactadherin (PAS 6/7) and Butyrophilin (BTN), have gained attention due to their antiviral, anticoagulant, anti-inflammatory properties, as well as the ability to control milk fat globule secretion. The protein aggregation and conformational changes that induced by temperature changes are important factors to determine the protein functionality. The objective is to study the temperature effects on buttermilk and the effect on physicochemical properties of PAS 6/7 and BTN. Cream obtained from local raw milk was churned in our laboratory and different heat treatments (4°C, LTLT, HTST, and 90°C for 10 s) were applied before and after churning to additionally assess the temperature effects on the proteins. This procedure was repeated by triplicate to assess variability. After each treatment, size exclusion methodologies were used to recover enriched protein extracts and dynamic light scattering analysis (DLS) was used to obtain particle size and the oligomeric state in solution. The zeta-potential was measured to determine charge changes. Fractionation of proteins by membrane filtration (100 and 30 kDa) showed the proteins suffered aggregation and interaction with whey proteins (SDS-PAGE); however, instead of associating with larger proteins under 4°C, whey proteins started to associate among themselves under HTST. To overcome this, gel filtration chromatography with Sephacryl S200 HR was used to maintain proteins native state. Before DLS and zeta-potential analysis, the isolated fraction was analyzed by native- and SDS-PAGE to identify PAS 6/7 and BTN, and assess the native oligomeric state and interaction with whey proteins. All results were analyzed by ANOVA. The results show the effect of temperature on MFGM, where maximum complexity was found in heating before churning, and indicate the need to better understand the effect of heat treatment on minor components of milk.