Fatty acids are potential trophic markers to trace feeding relationships in aquatic ecosystems primarily because lipid reserves of organisms broadly reflect dietary sources of lipids and can therefore provide information on the availability of key fatty acids in the food web. However, the use of fatty acids for such applications may be constrained by the degree to which the fatty acid composition of organisms is obscured by factors other than straightforward uptake from the diet. Thus, we studied the effect of long-term fasting, under controlled laboratory conditions, on the lipid content and fatty acid composition of field-caught Mysis relicta. Periods of 3 to 6 weeks are required to induce clear effects of fasting in M. relicta. Relative proportions of docosahexaenoic acid (DHA; 22:6n-3) and arachidonic acid (ARA; 20:4n-6) increased with decreasing lipid contents during fasting. DHA proportions in the total fatty acid composition of fasting M. relicta were significantly higher in comparison to field-caught animals. M. relicta with high proportions of DHA (> 25%) and with low lipid contents (< 14% of dry weight) can be clearly identified as fasting animals. Such thresholds will help to improve the validity of fatty acids in trophic studies.