A two-dimensional biomarker approach including fatty acids and stable isotopes of seston and copepods was applied to examine how the variability at the base of the food web affects trophic interactions between primary producers and copepod consumers over a sampling period of two years. We investigated how the composition of the seston affected feeding behaviour by analysing the fatty acid and stable isotope signals of the copepods Calanus helgolandicus, Acartia spp., Centropages spp. and Temora longicornis at Helgoland Roads, North Sea. Our results indicate that the relative contributions of autotrophic and heterotrophic fractions in the seston determined the stable isotope signal of the seston and hence the δ15N of copepods. Our findings show that the combination of stable isotope and fatty acid analyses provides an ideal tool to address the complexity of trophic relations in planktonic food-webs and to define relative trophic position and feeding preferences of e.g. copepods. Defining accurate baselines from bulk seston samples containing a mixture of auto- and heterotroph protist communities still remains a challenge when defining lower food-web dynamics in natural plankton communities.