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Background: Lumbar puncture (LP) is an invasive medical procedure that can be done by any doctor. Several simulation-based trainings have been built however the evaluations of the theoretical knowledge and the impact of the simulation-based training have never been performed in real life. The objective was to evaluate the impact of a LP training on the theoretical knowledge improvement and the performance of a LP in clinical practice., Methods: Before and after medical students' training, theoretical knowledge and confidence level were assessed. Over a 6 months period, the impact of simulation training was evaluated by the success rate of students' first LP carried out in hospitalized patients and compared to the results of a no-training control., Results: Students' theoretical knowledge and confidence level showed significant improvement after simulation training on 115 students (p < 0.0001). The evaluation in real life based on 41 students showed that the success rate of the first LP in patients was higher in the LP simulation group compared to the control group (67% vs 14%, p = 0.0025). The technical assistance was also less frequently needed in the LP simulation group (19% vs 57%, respectively, p = 0.017). The rate of students who participated in this educational study was low., Discussion: Simulation-based teaching was an effective way to improve students' theoretical and practical knowledge. Whether this approach translates to other procedural skills in real clinical settings merits further study. The low participation rate in the study is due to the fact that students are not used to be included in educational studies and to the complexity of evaluation in routine clinical practice.