Nutrient diffusing substrates (NDS) are an important tool for evaluating periphyton nutrient limitation. The rate at which nutrients are released from NDS depends on both the initial nutrient concentration and the length of time that NDS are in place. Whether temperature also affects nutrient release rates from NDS is unclear. However, this information is important because temperature effects on release rates could confound experimental results for NDS-based experiments testing rates of accumulation of periphyton biomass when stream water temperature is variable. We measured N and P release rates from NDS vials with 3 initial concentrations (0.05, 0.1, and 0.5 mol/L) of nutrients at 3 temperatures (4, 15, and 21°C) for 21 d. Release rates of both nutrients were greater for vials with higher nutrient concentrations and for vials at warmer temperatures. For all concentrations, release rates decreased log linearly with time, a result that might have important implications for patterns of colonization and subsequent interspecific interactions within the periphyton community. In our opinion, temperature-caused differences in release rates are not biologically important because the differences were much smaller (3%) than expected changes in periphyton maximum growth rates over similar temperature ranges (∼300%). Our results suggest that seasonal and site-related differences in temperature will not significantly affect nutrient release rates within the range of temperatures we tested, but researchers should consider nutrient concentration carefully when planning studies using NDS.