This paper examines the use of multi-stage or cascade ultrafiltration systems for continuous fractionation of model proteins human serum albumin (HSA) and human immunoglobulin G (HIgG). A three-stage, countercurrent ultrafiltration system was able to generate continuous HSA and HIgG streams with high purity and recovery. Pulsed sample injection ultrafiltration experiments showed that selectivity could be significantly affected by protein-protein interactions. The molar ratio of proteins in the feed was affected by the sieving coefficients of the proteins, particularly that of the preferentially transmitted one. Simulated results obtained with effective sieving coefficient data from pulsed sample injection ultrafiltration which factored in protein-protein interactions were found to be in good agreement with experimental results. The selectivity and hydraulic permeability as a function of time in a continuous fractionation process run under optimized conditions were determined to examine the feasibility of operation for extended duration. Crown Copyright (C) 2009 Published by Elsevier B.V. All rights reserved.