Humic substances (HS) possess redox active groups covering a wide range of potentials and are used by facultative anaerobic microorganisms as electron acceptors. To serve as suitable electron shuttles for anaerobic respiration, HS should be able to re-oxidize relatively quickly to prevent polarization of the surrounding medium. Mediated electrochemical oxidation and decolorization assays, based on the reduction of the radical ion of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS•−) allow to determine the electron donating capacity (EDC) of HS, but uncertainties remain about the reaction time that should be allowed to obtain environmentally meaningful EDC values. In this work, we performed a kinetic analysis of the time trend of the reduction of ABTS•− by HS by Vis and Electron Paramagnetic Resonance (EPR) spectroscopies and by cyclic voltammetry. We found evidences of two concomitant separate mechanisms of electron exchange: a fast and a slow transfer processes which may have different environmental roles. These results can set a base to identify the appropriate conditions for the spectrophotometric determination of the fast and slow components of the EDC of HS.