Resumo. Abstract Stable isotope analysis is a vital tool for elucidating nutrient flow within food chains. Studies utilizing this methodology must consider potential variables that could influence the δ13C and δ15N values of the species being examined. One such variable is the preservation of samples in a frozen state during field studies, particularly when immediate sample processing is not possible. Aim The objective of this study is to investigate the long-term effects of freeze-thaw at intervals of 1, 2, 7, 15, 30, 90 and 180 days on the δ13C, δ15N, and C:N ratios in biological samples from three fish species. Methods Tissues from three estuarine fish species (Cathorops spixii, Genidens barbus and Chloroscombrus chrysurus) were frozen and aliquots were subjected to thawing for sample collection aimed at isotopic determination. Results The discrepancies observed in this study were related to the δ13C and C:N ratios in the skin samples of C. spixii and G. barbus, as well as the muscle samples of C. chrysurus. These variations seem to be more closely linked to the potential volatility of the 12C isotope within the samples, resulting in δ13C enrichment and an increase in C:N values. Conclusions This suggests that these changes are not solely attributable to the freeze-thaw process, but may also be influenced by other factors like the lipid oxidation. Our findings suggest that this process significantly influences the δ13C and C:N ratios of skin and muscle tissues. Furthermore, we advocate for prompt processing of samples to minimize the impacts of freeze-thaw events.