We describe the total lipid content, lipid class composition and fatty acid profiles of adult forage fishes (anchovy, sardine and sprat) sampled in the NW Mediterranean Sea in 2010 and 2011. Inter- and intra-species differences were mostly related to sampling period with limited effect of gender or total length. As an assemblage, total lipid content and relative levels of triacylglycerols and fatty acids 16:1n7, 20:5n3 and 14:0 in forage fish were highest in summer and autumn, indicating better feeding conditions and a more pronounced diatom-supported food web. In contrast, total lipid content was lowest at the end of winter and spring, and coincided with high levels of 22:6n3, indicating a more herbivorous diet based on dinoflagellates. Resource partitioning and niche separation, as inferred from fatty acid profiles, were apparent between species. Sardine showed a more diverse, temporally separated feeding strategy than anchovy, and dietary overlap was higher in winter than summer with sardine having higher markers of copepods, 22:1n11 and 20:1n9. Sprat collected in winter occupied a separate niche area to both sardine and anchovy with higher total lipid content and carnivory biomarker 18:1n9. Our results show that the lipid dynamics of forage fishes can be used to gain quantitative insights into sub-system level changes in species interactions, including prey and predator productivity.