Diet is an important pathway for metal uptake in marine organisms, and assimilation efficiency (AE) is one of the most relevant parameter to quantify trophic transfer of metals along aquatic food webs. The most commonly used method to estimate this parameter is pulse-chase feeding using radiolabeled food. This approach is, however, based on several assumptions that are not always tested in experimental context. The present work aimed at validating the approach by assessing single-feeding and multiple-feeding approaches, using a model species (the turbot Scophthalmus maximus). Using the kinetic data obtained from the single-feeding experiment, we tested whether the reconstruction of a multi-feeding was consistent with data provided by an actual multi-feeding performed under the same experimental conditions. Our results validated the single-feeding approach.