Diel variability in nutrient concentrations is common but not universal in aquatic ecosystems. Theoretical models of photoautotrophic systems attribute the absence of diel uptake variation to nutrient scarcity, such that diel variability in nutrient uptake disappears as nutrients become limiting. We tested this prediction in a mesocosm experiment, by exposing benthic algal communities to a range of nitrogen (N) and phosphorus concentrations and recording the rates of uptake during both day and night. We found that higher concentrations of N produced diel variability in uptake and that the difference between the day and night total mass uptakes approximately equaled N demand for observed primary production as seen in other studies. At lower concentrations of N, uptake rates during the day and night were indistinguishable. These results are the first empirical evidence to imply that diel nitrate patterns in streams and rivers indicate a release from N limitation and offer a new way to assess nutrient limitation.