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In recent years, due to an increased need for non-intrusive sampling techniques, hydroacoustics has attracted attention in fishery science and management. Efforts to promote standardisation are increasing the accuracy, efficiency, and comparability of this method. The European Water Framework Directive and the Standard Operating Procedures for Fisheries Hydroacoustic Surveys in North American Great Lakes has recommended that surveys be conducted at night. At night, fish usually disperse in the water column, thus allowing for single echo detection and subsequent accurate fish size estimation, while day-time schooling behaviour hampers the estimation of fish size. However, sampling during the day would often be safer and cheaper. This study analyses how fisheries hydroacoustic results differ between day-time and night-time surveys, using data from 14 natural temperate lakes of various size. Data collected during the day and night at two depth layers linked to thermal stratification were compared in terms of acoustic scattering strength, target strength, and biomass estimates. The results showed a significant correlation between day-time and night-time estimates, though biomass in the upper layer was biased for day-time surveys, mainly due to incorrect fish size estimates resulting from rare single echo detections and schooling behaviour. Biomass estimates for the lower depth layer did not significantly differ between the two diel periods. Thus, this study confirms that hydroacoustic sampling in temperate lakes should be performed at night for accurate fish stock biomass estimates.