The deterioration of ion-exchange membrane (IEM) properties due to fouling and dissociation of water molecules at membrane-solution interface is considered to be an important limiting factor in electrodialysis (ED) separation processes such as ED with filtration membrane (EDFM). Non-stationary regimes such as pulsed electric field (PEF) is known to enhance ED efficiency by reducing fouling and water splitting, while polarity reversal (PR) of current with change in flow streams has been used as self-cleaning in place (CIP) tool in conventional ED processes. The present study aims to study, for the first time, the effect of PEF and PR (without changing the solution flow streams) on membrane characteristics and fouling behavior, energy consumption, and peptide migration rate as well as selectivity during peptide fractionation by EDFM. At the same time, the study was carried-out at two different constant voltages, 20 V (under-limiting current density) and 40 V (over-limiting current density). The peptide migration rate was unaffected for all types of electric field at 20 V while it was significantly lower with PR as compared to PEF and DC at 40 V. The selectivity of Arg and Lys was maximum in PEF mode at 20 V. A strong protective effect on membrane physicochemical properties was observed with PEF and PR regimes as compared to DC by reducing fouling and water dissociation at the membrane-solution interface, especially on AEM. Moreover, the relative amount of energy consumption was the lowest with PEF in relation to other two modes. Therefore, the present study demonstrated that PEF is energetically and technologically more feasible as compared to the conventional EDFM process with DC current. (C) 2016 Elsevier B.V. All rights reserved.