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Abstract RationaleStable isotope analysis (SIA) of free‐swimming mysticetes using biopsies is often limited in sample size and uses only one sample per individual, failing to capture both intra‐individual variability and the influence of demographic and physiological factors on isotope ratios. MethodsWe applied SIA of δ¹³C and δ¹⁵N to humpback whale (Megaptera novaeangliae) biopsies taken during the foraging season along the western Antarctic Peninsula to quantify intra‐individual variation from repeatedly sampled individuals, as well as to determine the effect of biopsy collection site, sex, and pregnancy on isotope ratios. ResultsThere was substantial variability in δ¹³C from multiple biopsies taken from the same individuals, though δ¹⁵N was much more consistent. Side of the body (left versus right) and biopsy location (dorsal, anterior, ventral, and posterior) did marginally affect the isotopic composition of δ¹⁵N but not δ¹³C. Pregnancy had a significant effect on both δ¹³C and δ¹⁵N, where pregnant females were depleted in both when compared to non‐pregnant females and males. ConclusionsThese results indicate that isotopic signatures are influenced by multiple endogenous and exogenous factors and emphasize value in accounting for intra‐individual variability and pregnancy status within a sampled population. Placed within an ecological context, the endogenous variability in δ¹³C observed here may be informative for future isotopic analyses.