Nickel-based catalysts reach a high activity for the hydrogen oxidation reaction (HOR) in anion exchange membrane fuel cells. While incorporation of iron significantly decreases the HOR overpotential on NiFe-based catalysts, the reason for the enhanced activity remains only partially understood. For the first time, in situ 57Fe Mössbauer spectroscopy is used to gain insights into the iron-related composition at different potentials. The aim is to evaluate which changes occur on iron at potentials relevant for the HOR on the active Ni sites. It is found that different pre-conditionings at low potentials stabilize the iron at a low oxidation state as compared to the as-prepared catalyst powder. It is likely that the lower average oxidation state enables a higher exchange current density and a more efficient OH adsorption, which make the Volmer step much faster in the HOR. Insights from in situ Mössbauer spectroscopy enlighten the role of iron in the nickel-iron catalyst, paving the way for developing improved Ni-based catalysts for HOR catalysis.