Display omitted •Simultaneous electroseparation of anionic and cationic peptides was studied by EDUF.•Migration rate increased with increasing peptide feed solution concentration.•Relative energy consumption decreased with increasing peptide concentration.•Peptide concentration affects the selectivity of free amino acids and/or peptides. The present work aimed to determine the effect of initial peptide concentration (0.5%, 1%, 2% and 4% (w/v) in feed solution during electrodialysis with ultrafiltration membranes (EDUF) on migration rate and selectivity of both anionic and cationic peptides as well as on electrodialytic parameters. The results showed that increasing the peptide concentration in the feed solution from 0.5% to 4% increased, in a linear way, the total separation rate and affected the selectivity of free amino acids in both recovery compartments RCA− (Asp, Glu and His) and RCC+ (Arg mainly) and/or peptides containing these amino acids. The highest migration rates observed at 4% were 16.2g/m2h and 7.8g/m2h for the cationic and anionic compartments respectively. Furthermore, the relative energy consumption decreased with increasing feed solution concentration from 17.4Wh/g at 0.5% concentration to 3.53Wh/g at 4% concentration. In addition, membrane integrity and physicochemical properties were not affected in the range of peptide concentration tested. To the best of our knowledge, it was the first time that the impact of feed peptide concentration on migration, selectivity and relative energy consumption is demonstrated in EDUF.