Our experimental study was designed to assess the effects of temperature on nitrogen isotope turnover and to measure the effects of temperature and food quality on the stable carbon and nitrogen isotope discrimination factors (Δ13C and Δ15N) in a cladoceran. For the first part of our study, Daphnia were fed with non-enriched or 15N-enriched Scenedesmus obliquus at 12, 15, 20, and 25 °C. For the second part, Daphnia were reared at 15, 20, and 25 °C on Scenedesmus or Cryptomonas sp. There were no clear effects of temperature on turnover rates of the nitrogen isotope of cladocerans. However, a general increase in Δ13C with increasing temperature was measured, regardless of the food source. Δ15N was also affected by temperature, but contrasting results were measured depending on the food source used. There were significant effects of food quality on Δ13C and Δ15N in Daphnia, as values obtained for Daphnia fed Scenedesmus were always higher than those obtained for Daphnia fed Cryptomonas. Our experiments produced discrimination factors that were very different from those usually considered in isotope studies and showed that the values used for isotope model implementation to analyze field data need to be adapted to environmental conditions.