The aim of this study was to investigate the influence of membrane pore size and filtration temperature on six individual milk protein fractions (αS-CN, β- CN, κ-CN, α-LA, β-LG A, β-LG A) during the protein fractionation microfiltration process. Pasteurised skimmed milk was microfiltrated using two different pore sizes of spiral-wound membranes, with pore sizes of 0.2 μm and 0.5 μm, at temperatures of 15 °C and 45 °C respectively. The microfiltration process was carried out with a final volume reduction of 66% and a diafiltration volume of 120% (300 L) of the original feed (250 L). It was observed that neither the pore size nor the filtration temperature significantly (p < 0.05) affected the permeation of the α-LA fraction. However, the permeation of the β-LG A and β-LG B fractions can be influenced by membrane pore size and filtration temperature, and the behaviour of the three whey protein fractions, A and B genetic variants of the β-LG and α-LA fractions differs significantly during the microfiltration process. The results of this study could form the basis for the development of new, unique tailor-made milk protein ingredients.