A literature survey of zooplankton stable isotope studies revealed inconsistencies between authors concerning (a) fixation and (b) allowance for gut clearance of zooplankton prior to δ13C and δ15N determinations. To address whether commonly used preservation techniques induce changes in stable isotope values, fresh lake zooplankton (control) were compared with preserved (ethanol, methanol, formaldehyde, gluteraldehyde, frozen and shock frozen) material. Differences of up to 1.1‰ for carbon and 1.5‰ for nitrogen isotopic signatures were found. Even freezing, the most frequently used method identified from the literature, caused significant changes compared with the control. We advocate the use of fresh material prepared immediately whenever possible, or complementary testing of the preservative method to be used. Larger organisms are routinely eviscerated, or specific tissues are dissected, and analysed for stable isotopes to reduce errors introduced via the gut contents. Yet zooplankton gut clearance is rarely performed: the gut content assumed to be negligible relative to organism mass. Experimental determinations of relative gut mass, from both original and compiled data, range from 1–26% for different zooplankton species. Using reported isotopic values of basal resources from natural systems, we calculated that, when analysing bulk zooplankton, inclusion of the gut mass may introduce substantial errors of >3‰. Thus it appears prudent to perform the simple procedure of gut clearance, especially for copepod species.