The ecologies of vertebrate species have been increasingly studied via stable isotope analyses of small quantities of body tissues. However, critical assumptions relating to the consistency in stable isotopic values in consumer tissues and their diet as well as the rate of incorporation of diet-derived stable isotopes into consumer tissues remain poorly validated for most taxa despite numerous stable isotope studies targeting natural systems. In this study, we measured stable carbon and nitrogen diet–tissue discrimination and elemental turnover in whole blood, red blood cells, blood plasma, brain, liver, pectoralis major muscle, pubioshiofemoralis internus muscle, stratum corneum (sc) dermis, and stratum germinativum (sg) dermis of Pond Sliders (Trachemys scripta) fed ad libitum on two isotopically distinct diets. Turtles were fed a soy-based control diet (32% protein, 3% lipids, 4% fiber), and after 146 d, a subset was switched to a fish meal-based experimental diet (44% protein, 24% lipids, 3% fiber). At the diet switch, diet–tissue isotopic equilibrium could not be confirmed by δ¹³C values for any tissue, but was established for δ¹⁵N values for all tissues except sc dermis and sg dermis (range  =  1.9 to 3.8‰). An exponential-decay regression model and half-life calculation resulted in half-lives for N ranging from 35.6 d (blood plasma) to 52.5 d (liver). These represent the first published stable isotope turnover rates for reptiles and the first diet–tissue isotopic discrimination factors for freshwater turtles. The discrimination factors found here are similar to those established for soft-tissues among other vertebrate taxa; however, elemental nitrogen turnover rates in tissues of T. scripta are among the slowest established to date among vertebrate taxa.