Nitrate contamination is a common problem in groundwater around the world. Nitrate can be cathodically reduced in bioelectrochemical systems using autotrophic denitrifiers with low energy investment and without chemical addition. Successful denitrification was demonstrated in previous studies in both microbial fuel cells and microbial electrolysis cells (MECs) with continuous current flow, whereas the impact of intermittent current supply (e.g., in a fluidized-bed system) on denitrification and particularly the electron-storing capacity of the denitrifying electroactive biofilms (EABs) on the cathodes have not been studied in depth. In this study, two continuously fed MECs were operated in parallel under continuous and periodic polarization modes over 280 days, respectively. Under continuous polarization, the maximum denitrification rate reached 233 g NO3–-N/m3/d with 98% nitrate removal (0.6 mg NO3–-N/L in the effluent) with negligible intermediate production, while under a 30 s open-circuit/30 s polarization mode, 86% of nitrate was removed at a maximum rate of 205 g NO3–-N/m3/d (4.5 mg NO3–-N/L in the effluent) with higher N2O production (6.6–9.3 mg N/L in the effluent). Conversely, periodic polarization could be an interesting approach in other bioelectrochemical processes if the generation of chemical intermediates (partially reduced or oxidized) should be favored. Similar microbial communities dominated byGallionellaceaewere found in both MECs; however, swapping the polarization modes and the electrochemical analyses suggested that the periodically polarized EABs probably developed a higher ability for electron storage and transfer, which supported the direct electron transfer pathway in discontinuous operation or fluidized biocathodes.