Isotopes are increasingly used in rainfall-runoff models to constrain conceptualisations of internal catchment functioning and reduce model uncertainty. However, there is little guidance on how much tracer data is required to adequately do this, and different studies use data from different sampling strategies. Here, we used a 7-year time series of daily stable water isotope samples of precipitation and streamflow to derive a range of typical stream sampling regimes and investigate how this impacts calibration of a semi-distributed tracer-aided model in terms of flow, deuterium and flux age simulations. Over the 7 years weekly sampling facilitated an almost identical model performance as daily, and there were only slight deteriorations in performance for fortnightly sampling. Monthly sampling resulted in poorer deuterium simulations and greater uncertainty in the derived parameter sets ability to accurately represent catchment functioning, evidenced by unrealistic reductions in the volumes of water available for mixing in the saturation area causing simulated water age decreases. Reducing sampling effort and restricting data collection to 3 years caused reductions in the accuracy of deuterium simulation, though the deterioration did not occur if sampling continued for 5 years. Analysis was also undertaken to consider the effects of reduced sampling effort over the driest and wettest hydrological years to evaluate effects of more extreme conditions. This showed that the model was particularly sensitive to changes in sampling during dry conditions, when the catchment hydrological response is most non-linear. Across all dataset durations, sampling in relation to flow conditions, rather than time, revealed that samples collected at flows >Q50 could provide calibration results comparable to daily sampling. Targeting only extreme high flows resulted in poor deuterium and low flow simulations. This study suggests sufficient characterization of catchment functioning can be obtained through reduced sampling effort over longer timescales and the targeting of flows >Q50.