Microfiltration, MF is largely used in dairy industry to separate casein micelles from whey proteins. The weak performances of polymeric membranes compared to ceramic membranes are a restriction for their implementation in industry. Adding a diafiltration step is promising to improve polymeric MF performances, however most of the works have focused on the hydraulic performances and few have investigated the repercussion of diafiltration on protein separation. This work aims at investigating the impact of both the nature of solvent and the diavolume ratio on the MF efficiency. Pilot-scale MF using polymeric membrane (Synder, 800 kDa) was carried out on thermized skimmed milk at 12 °C with a volume reduction ratio, VRR from 2.5 to 3.2 and a transmembrane pressure of 0.7 bar. Continuous diafiltration was performed using reverse osmosis water (ROW) or milk ultrafiltrate (PUF). Permeation flux and β-lactoglobulin transmission were evaluated. During PUF-diafiltration of VRR-2.8-retentate, permeation flux is stable and β- lactoglobulin transmission declines by 26 % after 4.8 diavolumes. During ROW-diafiltration, permeation flux increases linearly until 2.9 diavolumes, then reaching an upper value. Simultaneously β-lactoglobulin transmission follows a parabolic curve with a maximum increase of 68 % for VRR-3.2-retentate and 10 % for VRR-2.5-retentate. According to Darcy’s law, the reduction of permeate viscosity during ROW-diafiltration leads to the improvement of permeation flux. This behavor is accentuated by the fouling layer relaxation consequent to the diminution of the ionic strength. In case of high repulsive forces in the concentration layer (low ionic strength), the transmission of negatively charged whey proteins is however restricted. The decrease of protein concentration during diafiltration may also limit whey protein transmission for high diavolume ratios. Diafiltration using ROW is recommended to increase transmission of whey proteins. However, volume of solvent has to be adapted to the VRR reached prior diafiltration to enable an optimum transmission