The opossum shrimp Mysis relicta is an important component of the diet of benthivorous and planktivorous fish in the Great Lakes. The invasion of the Great Lakes by exotic invertebrates (Bythotrephes longimanus, Cercopagis pengoi, Dreissena polymorpha, and D. bugensis) has altered the base and intermediate levels of the foodweb. Thus, information about the condition of M. relicta may reveal the extent of indirect effects of these changes on this trophically-important invertebrate. Biochemical indices based on nucleic acid ratios have been shown to be suitable proxies for the growth and condition of aquatic organisms. These indices are affected by multiple factors, such as; food level, temperature, body size, sex/life stage, maturation, and moult stage and need to be calibrated before field data can be interpreted on a quantitative basis. In this study, we investigated the effect of fasting under different temperature conditions on the nucleic acid ratios RNA/DNA, RNA/protein and protein/DNA in M. relicta. Juvenile M. relicta were exposed to fasting conditions for 11 and 21 d in two controlled laboratory experiments at 3°C and 8°C. Several effects of time and temperature on the condition indices of fasting M. relicta were observed; however, we concluded that, of the various metrics tested, only RNA/DNA ratios provide a suitable index of metabolism and condition in fasting animals. RNA concentrations declined in response to fasting on the order of 3–4 d at 8°C and between 4 and 11 d at 3°C. Juvenile M. relicta with RNA/DNA ratios < 1.5–1.8 were clearly identified as fasting animals. Field-caught animals having RNA/DNA ratios near these levels are demonstrating clear signs of metabolic stress.