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The influences of river regulations on aquatic biota are insufficiently investigated. We assessed the level of ecological disturbance of the mayfly assemblages through the damming and flow alterations at two highly regulated Mediterranean rivers in Croatia: the Cetina River and its tributary, the Ruda. Contrary to our hypotheses, species richness, abundance and diversity of mayflies increased along the river’s course. At the sites directly influenced by the river regulations, mayfly assemblage changed: it was less diverse and less abundant. However, no obvious influence of the river regulation on the mayfly assemblages was detected for the sites that were indirectly influenced by these alterations. This was most likely due to specific karst hydrology, i.e., the presence of lateral springs along the course of the river. Multidimensional scaling analysis (MDS) revealed grouping of sites according to the similarity in physico …