Surplus electricity may be supplied to anaerobic digesters as H-2 gas to upgrade the CH4 content of biogas. Acetate accumulation has been observed following H-2 injections, but the parameters determining the degree of acetate accumulation are not well understood. The pathways involved during H-2 consumption and acetate kinetics were evaluated in continuous lab reactors and parallel batch C-13 experiments. Acetate accumulation increased during initial H-2 injections as organic loading rate increased and CO2 levels decreased below 7%. The share of CH4 in H-2 and C-13 mass balances increased after repeated H-2 injections, which corresponded with the increase of Methanomicrobiales observed via qPCR. The organic loading rate, the inorganic carbon level and level of methanogen adaption hence determine acetate kinetics during biomethanation of H-2. The three identified parameters may form the base of a decision tool to assess acetate accumulation during H-2 injections to an anaerobic digester.