Flow regulation via impoundments threatens lotic ecosystems and the services they provide globally. Impoundments drastically alter flow and stream temperature variability within fluvial environments, but efforts to quantify ecohydrological and ecothermal responses to flow regulation in conjunction have been sparsely explored to date. This study examined macroinvertebrate community responses to antecedent flow (discharge) and stream temperature variability across paired regulated and non-regulated systems associated with three reservoirs located in adjacent catchments. Community abundances, functional traits, and biomonitoring indices were examined, and ecological differences between non-regulated and regulated sites were quantified, with the most sensitive faunal response being correlated against a suite of flow and thermal indices.Regulated sites exhibited reduced low-flow variability and rapid increases in discharge during peak flows that regularly exceeded those conveyed by non-regulated sites, while stream temperature variability was highly congruent between sites. Macroinvertebrate functional traits were particularly sensitive to flow regulation, and incorporating biomonitoring indices marginally improved the ecological discrimination between regulated and non-regulated sites. Unlike community abundances, functional traits did not vary spatially between catchments, highlighting that such information could guide the implementation of regional environmental flows.Macroinvertebrate communities responded significantly to various hydrological parameters, particularly those associated with the timing of extreme flows, but were less sensitive to thermal controls. Future research should explore ecological responses to antecedent hydrological and stream temperature variability associated with flow regulation to provide a better understanding of the underlying mechanisms driving biotic alterations, which could guide future environmental flow methodologies.

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