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Background: Shorter door-to-needle time (DNT) essentially contributes to the effectiveness of thrombolytic treatment. Simulation-based training could shorten DNTs. Here we present the first results of simulation training, designed to shorten DNT ≤ 20 minutes, conducted in the Czech Republic.Methods: The simulation-training scenario was developed and run by the Comprehensive Stroke Center of St. Anne’s University Hospital in Brno. It has a standard format (intro-simulation-debriefing-conclusion) and includes two rounds of simulation, as well as a video presentation of real thrombolytic cases. Between June 2016 and March 2017, St. Anne’s Hospital staff received this training, followed by 4 of the 5 closest primary stroke centers. The DNTs of hospitals which received training were compared to the DNT of all the other stroke centers in the Czech Republic in the 4-month period pre-training (March - June 2016), as well as post-training (April - July 2017), using data from the SITS registry.Results: In 5 centers participating in simulation training, DNT was 47 minutes (95%CI 45-52; 102 patients) pre-, and 29 minutes (95%CI 24-38; 88 patients) post-training. In 45 stroke centers without training, DNT was 40 minutes (95%CI 40-41; 860 patients) before and 30 minutes (95%CI 28-31; 421 patients) after the simulation period. In trained stroke centers, the percentage of patients that achieved DNT ≤ 20 minutes was 3% pre-training, and increased to 32% post-training (OR=15, 95%CI 4.5-53). In all other stroke centers, 11% and 27% of patients were thrombolyzed with DNT ≤ 20 minutes before and after the simulation period, respectively (OR=3, 95%CI 2.1-4.0).Conclusion: DNT was shortened in all stroke centers. Additionally, training further improved logistics and increased the percentage of patients thrombolyzed within 20 minutes. Simulation training is a powerful tool for improving hospital logistics.