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The pandemic of coronavirus disease (COVID-19) and related restrictions (closed schools and sports centers, social isolation, masks) may have a negative impact on children's health. The purpose of this study was to evaluate the level of physical activity (PA) of Czech children during COVID-19 in autumn 2020. Ninety-eight Czech children (mean age = 10.1 ± 1.47 years) completed the standardized Physical Activity Questionnaire for Older Czech Children (PAQ-C/cz) during COVID lockdown. Data were compared with previously published norms. Thirty-five children also reported daily number of steps measured by accelerometers. Total PAQ-C score was 0.38 lower during COVID compared to Pre-COVID [t(302) = 5.118., p < .001]. The male PAQ-C total score was 0.37 lower [t(146) = 3.21., p = .002)] and the female total score was 0.39 lower [t(154) = 3.97., p < .001] during COVID compared to Pre-COVID. Specifically, responses of PA during spare time, before-school, physical education (PE), and recess were significantly lower during COVID. The average number of steps was 7.767 steps/day (boys = 9.255; girls = 6.982). COVID lockdown resulted in significant reduction of PA in Czech children. Strategies to promote adequate PA of children during the pandemic need to be determined.