Stable isotope data are used to track diets and movements of fish. Reliable interpretation depends on understanding fractionation between diets and tissues of consumer fish. We experimentally investigated the influence of diet on fractionation of carbon and nitrogen isotopes in mummichog, Fundulus heteroclitus, a common estuarine species in the eastern US. Fish muscle was analyzed both with and without lipids depleted in order to examine variability in isotopes that may be associated with muscle lipid content. Fish that had their diets swapped from artemia to either squid, clam or one of two artificial diets had uniquely different muscle isotopic composition than fish reared with a constant artemia diet. For all treatments, fish muscle was enriched in carbon and nitrogen isotopes compared to their diets. Lipid-depleted muscle was on average 1 per mil enriched compared to non-depleted muscle, and within and between treatment variability was not greatly altered. Our results show that assumptions of constant isotopic enrichment with respect to dietary sources will not always apply to ecological studies.