AbstractRiver networks exhibit a globally important capacity to retain and process nitrogen. However direct measurement of in‐stream removal in higher order streams and rivers has been extremely limited. The recent advent of automated sensors has allowed high frequency measurements, and the development of new passive methods of quantifying nitrogen uptake which are scalable across river size. Here we extend these methods to higher order streams with anthropogenically elevated nitrogen levels, substantial tributaries, complex input signals, and multiple N species. We use a combination of two station time‐series and longitudinal profiling of nitrate to assess differences in nitrogen processing dynamics in a natural versus a channelized impounded reach with WWTP effluent impacted water chemistry. Our results suggest that net mass removal rates of nitrate were markedly higher in the unmodified reach. Additionally, seasonal variations in temperature and insolation affected the relative contribution of assimilatory versus dissimilatory uptake processes, with the latter exhibiting a stronger positive dependence on temperature. From a methodological perspective, we demonstrate that a mass balance approach based on high frequency data can be useful in deriving quantitative uptake estimates, even under dynamic inputs and lateral tributary inflow. However, uncertainty in diffuse groundwater inputs and more importantly the effects of alternative nitrogen species, in this case ammonium, pose considerable challenges to this method.