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The management of environmental flows is of paramount importance in regulated water resources systems to preserve river ecosystems. This work proposes a methodology to assess habitat alteration in river basins altered by management activities. The methodology is based on the joint application of a basin management model (SIMGES, AQUATOOL) and a model to estimate habitat time series (CAUDECO). CAUDECO is based on the weighted useable areas of the species in their different vital stages that, in turn, depend on the flows in each river stretch and the biological periods of the species. The final output is an indicator of habitat alteration, which is defined ad hoc for this work to relate the habitat suitability under regulated and natural regimes. The methodology was applied to a case study in north-western Spain: the Órbigo River basin. The results in the current management scenario highlight that the ecological flows improve the habitat suitability of several species with respect to natural regime conditions. For instance, the mean values of the habitat time series in the Órbigo River for the brown trout and bermejuela under regulated conditions are 69.6% and 88%; whereas in natural regime they are equal to 55.1% and 72.9%, respectively. Based on these results, eight additional scenarios of ecological flows were tested and their effects on both habitat alteration and water demand reliability were quantified and discussed. It was found that increases in the ecological flows up to 30% do not affect the reliability of water demands and reduce habitat alteration (i.e., lead to values of the habitat alteration indicator closer to 1) for all species present in the river basin. These results highlight that the methodology and indicator of habitat alteration proposed in this paper are useful to support the management of regulated river basins, since they allow assessing the implications of ecological flows on both habitat suitability and reliability of water demands. • A methodology is proposed to assess habitat alteration in regulated basins. • The methodology is applied to the Órbigo River basin (north-western Spain). • Environmental flows can improve habitat suitability compared to natural conditions. • Increases in environmental flows reduce habitat alteration for all species. • Rises in environmental flows up to 30% do not affect the reliability of water demands. [ABSTRACT FROM AUTHOR]