Cold skimmed milk microfiltration using polymeric spiral-wound membranes has a growing interest to separate serum proteins from casein micelles. Diafiltration using reverse osmosis water (ROW-DF) is needed to increase the transmission of serum proteins. The objective of this study was to investigate the performance of skimmed milk microfiltration in ROW-DF mode when operating with single industrially sized standard SW modules at low temperature (~12 • C). As expected during ROW-DF, the permeation flux increased with the rate of diafiltration, due to both the decrease of permeate viscosity and decrease of ionic strength. It is shown for the first time, that the transmission of protein (mainly β-Lactoglobulin, β-LG, major serum protein of bovine milk) presents different scenarios (increase, decrease and 'parabolic' shape) as function of diafiltration rate and operating conditions. These performances are quite different from the ones obtained when operating diafiltration with permeate from milk ultrafiltration, PUF-DF, and can be explained by the modifications induced by ROW on the properties of the fluid (feed) and on the properties of the concentrated layers of casein micelles accumulated at the membrane. The quantity of β-LG which can be released into the permeate was linked to the concentration factor applied before ROW-DF and the ability of deposit to swell was linked to the transmembrane pressure applied as critical conditions could lead to a cohesive deposit unable to swell. If the increase of repulsive electrostatic interactions (induced by the increase of diafiltration rate) between casein micelles and β-LG supported the release of β-LG entrapped in the deposit, it impaired the crossing of β-LG remaining in the retentate. The result was a progressive decrease of β-LG transmission with the increasing diafiltration rate. In summary, transmission of β-LG was maximized for low value of transmembrane pressure (0.7 bar), a high value of concentration factor (3.2) and an intermediate diafiltration rate (2.1).