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Urban heat islands (UHI) are a well-known phenomenon adversely affecting human health and urban environments. The worldwide COVID-19 lockdown in 2020 provided a unique opportunity to investigate the effects of decreased emission of air pollution and anthropogenic heat flux (AHF) on UHI. Although studies have suggested that reduced AHF during lockdown decreased atmospheric UHI (AUHI) and surface UHI (SUHI), these results contain inherent uncertainties due to unaccounted weather variability and urban-rural dynamics. Our study comprehensively analyzes the impact of the COVID-19 lockdown on AUHI and SUHI in Prague, Czechia. By selecting days with similar weather conditions, we examined changes in mean SUHI using MODIS satellite images and in AUHI based on air temperature from Prague weather stations for the Lockdown period during March–April 2020 versus a Reference period from March–April 2017–2019. Our results show that, in comparison to the Reference period, the Lockdown period was associated with a 15% (0.1 °C) reduction of SUHI in urbanized areas of Prague and a 0.7 °C decline in AUHI in the city center. Additionally, the observed decreases in satellite-based aerosol optical depth and nitrogen dioxide by 12% and 29%, respectively, support our hypothesis that the weakened UHI effects were linked to reduction in anthropogenic activities during the lockdown. Revealing the largest decrease of mean SUHI magnitude around the periphery of Prague, which has predominantly rural land cover, our study emphasizes the need to consider the effects of urban-rural dynamics when attributing changes in SUHI to AHF. Our findings provide additional insights into the role of reduced anthropogenic activities in UHI dynamics during the COVID-19 lockdown and offer policymakers a comprehensive understanding of how the complex interaction between urban and rural microclimate dynamics influences the SUHI phenomenon. [ABSTRACT FROM AUTHOR]