We used high‐frequency in situ measurements of nitrate (NO3−) and dissolved oxygen (DO) from the springfed Ichetucknee River, Florida, to derive multiple independent estimates of assimilatory nitrogen (N) demand, and to evaluate the short‐term dependence of heterotrophic assimilation and dissimilation (e.g., denitrification) on gross primary productivity (GPP). Autotrophic N assimilation estimates derived from diel DO variability and GPP stoichiometry agreed closely with estimates based on integration of diel variation in NO3− concentration, although the correspondence of these metrics depended on the method used to estimate NO3− baselines. In addition, day‐to‐day changes in nocturnal NO3− concentration maxima were strongly negatively correlated with day‐to‐day changes in GPP. Diel temperature variation in the Ichetucknee River indicated that this pattern could not be explained by hydrologic dispersion, while relationships between N assimilation and O2 production at hourly intervals indicated minimal physiological lags. The estimated magnitude of heterotrophic assimilation was small, indicating that the relationship between changes in GPP and changes in nocturnal NO3− maxima reflects sensitivity of denitrification to variation in exudation of labile organic matter by primary producers. We estimate that ~ 35% of denitrification may be fueled by the previous day’s photosynthesis; this result is consistent with the broader hypothesis that the magnitude of autochthonous production in aquatic systems influences the fate of N via both direct and indirect mechanisms.