This study focuses on the importance of the feed volume as an operational parameter for improving the performance of a cyclically fed glycerol-based microbial fuel cell. Experiments varying the daily feed volume from 10 to 50 mL for a 240 mL anode evidences that the electrochemical performance, Chemical Oxygen Demand (COD) and nitrogen and phosphorous removal is affected, in addition to the Coulombic efficiency (CE) and the kinetics of the glycerol biodegradation. A feed volume of 32 mL (hydraulic retention time (HRT) of 7.5 d) is shown to be the most effective for maximising the cell performance and COD removal. Longer HRTs reduce the organic loading rate, thereby reducing the cell performance due to the rapid depletion of the substrate, whereas shorter HRTs favor the development of non-exoelectrogenous bacteria, which reduces the CE and electrochemical performance with less effective COD removal. A final product analysis reveals that formic acid is the main product formed as a consequence of the microbiological degradation of glycerol.