In an electrodialysis with filtration membrane (EDFM) separation process, a pair of restriction membranes is used to prevent leakage of molecules into the electrode solution. Driven by the electrical potential gradient, ions transport across these membranes in different directions, resulting in concentration changes in the feed and permeate compartments respectively. However, the effects of these restriction membranes have not been systemically studied to date. In the present work, the performance of a non-charged polyacrylamide (PAm) membrane and two ion-exchange membranes were compared. PAm restriction membranes were found to give the most stable operation, with only slight concentration and pH changes in the feed and permeate compartments. The configurations with either cation or anion exchange membranes showed more significant changes in electrolyte concentration in opposing directions but the same pH trend, as the current density increased. The configuration with cation exchange membranes as restriction membranes resulted in the highest protein recovery, while the use of anion exchange membranes would appear as the best choice for separation of a protein with net positive charge. The results of this work provide important insights for the design and operation of EDFM processes and a better understanding of the transport phenomena that occur during the separation of bioactive compounds.