The aim of this study was to evaluate changes in AV nodal properties during administration of metoprolol, using a novel ECG-based method for parameter estimation. The AV nodal parameters account for the probability of an impulse not passing through the fast pathway, the absolute refractory periods of the slow and fast pathways (aRPs and aRPf), representing the functional refractory period, and related prolongation in the respective refractory periods. Twenty patients (age 71±8 years, 14 men) with permanent AF from the RATe control in Atrial Fibrillation (RATAF) database were included in this study. Recordings during baseline and metoprolol administration were analyzed. Furthermore, simulated RR series were generated mimicking metoprolol administration. During metoprolol administration, aRP was significantly prolonged in both pathways (aRPs: 342±39 vs. 408±81 ms, p<0.001; aRPf: 432±74 vs. 527±83 ms, p<0.001). Similar results were found for the simulated RR series: both aRPs and aRPf were significantly prolonged with metoprolol. The AV nodal parameters reflect expected changes after metoprolol administration, i.e., a prolongation in functional refractory period. The simulations suggest that aRP may serve as an estimate of the functional refractory period.