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Hydropeaking leads to major anthropogenic disturbance of river networks worldwide. Flow variations imposed by hydropeaking may significantly affect macroinvertebrate assemblages within the river network. As such, the responses of macroinvertebrate assemblages to hydropeaking are expected to be complex and vary across spatial and temporal scales as well as ecological organization levels. To unpack this complexity, we assessed the interplay of geomorphic and hydrological variables as drivers of the responses of macroinvertebrate assemblages to hydropeaking. Specifically, we studied different levels of ecological organization of macroinvertebrate assemblages in two functional process zones (FPZs; Sub-Andean and Central Valley Gravel Dominated) subjected to different flow management in two Chilean Andean river networks. Hydropeaking caused significant reduction of macroinvertebrate abundances in both FPZs and at all ecological organization levels with the exception of one feeding guild (scrapers). Furthermore, the response of macroinvertebrate assemblage variance was stronger in the Central Valley Gravel Dominated FPZ. Both geomorphic and hydrological variables influenced macroinvertebrate assemblage responses. However, the effects of the principal geomorphic variables operated at valley (meso) spatial scale and the main hydrological variables operated at the sub-daily (micro) temporal scale. Therefore, to minimise the effects of hydropeaking on macroinvertebrate assemblages, flow management should consider reduction of sub-daily variability. Furthermore, placement of new barriers should take into account not only their position within the river network but also their effects downstream that strongly depend on characteristics of river valley., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)