AbstractExploring the contributions of new and old water to runoff during precipitation events in agricultural catchments is essential for understanding runoff generation, solute transport, and soil erosion. The aim of this study was to investigate the variability in the isotopic composition of precipitation and runoff in the 66 ha agricultural catchment in Austria, in the Hydrological Open Air Laboratory (HOAL), in order to compare two isotope hydrograph separation methods. The classical two‐component (IHS) and the ensemble hydrograph separation (EHS) were applied to multiple large events in May–October of 2013–2018 using δ18O and δ2H. The peak flow new water contributions obtained by IHS were compared with the average new water fraction from EHS. The average new water fraction calculated with EHS based on regular weekly sampling was close to zero, which can be explained by the large diffuse groundwater discharge into the stream between the events. When only investigating events with high temporal resolution sampling, the results suggest that EHS provided average new water fractions during peak flows (0.46 ± 0.04 for δ18O, 0.47 ± 0.03 for δ2H) that were close to the averages obtained by IHS (0.47 for δ18O, 0.50 for δ2H). New water fractions tended to be higher for larger rainfall intensities. High peak flow new water fractions could be explained by the agricultural land use and soils with low permeability promoting overland flow generation and by some of the tile drainage systems contributing to the delivery of water. In conclusion, a weekly sampling frequency was not sufficient in the HOAL but instead high‐resolution sampling during events was necessary to estimate the average new water contributions during events. While EHS may be a more robust approach compared to IHS, as it relaxes some of the assumptions of IHS, IHS can provide information on the variability of new water contributions of individual events.