A hybrid membrane cell comprising of semi and fully-permeable membrane sections, mounted in series, was employed to study the influence of variable transport properties (diffusivity and viscosity) in the separation of two macromolecules by a differential diffusivity mechanism. Separation pairs of bovine serum albumin (BSA)/dextran-T10 (DT10) and bovine serum albumin (BSA)/lysosyme (LYS) were considered. A numerical code was developed to quantify the effect of variable transport properties in the concentration, permeate velocity, and purity of the outgoing streams. The variation of the transport properties with concentration reinforces the accumulation of solutes over the semi-permeable membrane. The separation is more effective, with a higher concentration of BSA in the stream crossing the fully permeable membrane, when the accumulation of BSA is reinforced, comparatively to that of the other solute, and vice-versa. For high concentration polarization at semi-permeable membranes surface, the ratio between concentrations of BSA/DT10 and BSA/LYS is equal to 2.0 and 1.4, respectively. BSA purity in the outgoing stream reaches 60% and 56.5% for BSA/DT10 and BSA/LYS, respectively, compared to the 50% at the cell inlet. For the same operating conditions, supposing constant transport properties, the differential of purity is 20% lower for BSA/DT10 system and 6% higher for BSA/LYS.