Tissue-diet discrimination factors (TDDFs) for carbon (∆δ13C) and nitrogen (∆δ15N) were estimated for two brittle star species (Ophiocomina nigra and Ophiothrix fragilis) in tissues of two body compartments (disk and arms) according to different diets (fish muscle, mussel and macroalgae). Variations in TDDFs are studied in the light of physiological changes, as two phases were revealed over the course of the experiment, i.e. a gonadal rest and a gonadal maturation. Overall, the average ∆δ13C and ∆δ15N values ranged from − 4.1 ± 0.2 to 1.5 ± 0.2‰ and from − 1.8 ± 0.2 to 4.0 ± 0.3‰, respectively. Variability in TDDFs revealed little differences between species but appeared to be mainly driven by the diet and the physiological state of the organisms to a lesser extent. ∆δ13C in gonadal resting organisms were systematically ca. 0.8‰ lower than in gonadal maturing species. TDDF values for organisms fell within the range of expected values, except for ∆δ13C with macroalgae diet and ∆δ15N with fish muscle diet. As revealed by %N and CN ratios, high dietary-protein seemed to play a key role in explaining ∆δ15N values, while ∆δ13C seemed to be affected by differential assimilation efficiency in dietary components. We also suggested a possible effect of isotopic signature of the diet on TDDFs showing that the more enriched the diet in δ15N and δ13C, the lower the TDDF.