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Background Simulation has been recognized as a shift in health care education that can improve skills and patient safety and outcomes. High-fidelity simulation of critical medical situations can be a source of stress among participants that can interferes with students' abilities leading to unexpected emotional response. In the current study, we sought to evaluate stress responses during a session including a high-fidelity simulation dedicated to the management of an emergency and one performing a complex technique such as spinal tap, in a population of medical students during a session including high-fidelity simulation (HF) dedicated to the management of an emergency and one performing a complex technique such as spinal tap (ST). Methods A quasi-experimental before-after study was used including the administration of questionnaires before and after simulation, and biomarkers evaluation by salivary cortisol samples. A total of 148 students 148 were eligible and agreed to participate in the study. We used paired T-test for mean comparison regarding State-trait anxiety for both HF and ST simulations. For NASA-TLX we performed a T-test mean comparison between groups. We used paired T-test mean comparison for cortisol analysis in the HF simulation. Multivariable linear regression has been used to assess variables associated with AUCg Results values of State-Trait Anxiety Inventory scores were relatively higher at the end of the HF and ST simulation sessions. NASA task load index (NASA-TLX) was significantly higher at baseline for the high fidelity simulations, with respect to the ST simulation. Regarding HF simulation, a T-test for paired samples showed a statically significant difference between cortisol levels at 10 minutes after the start of the training and after 30 minutes from the simulation in both groups. Fold increase was similar in the two groups. Linear regression showed that cortisol AUCg was related to the STAI-Y score in both groups. Conclusions This work highlights how our students developed a stress response, testified by psychological and biological outputs more related to their personal attitude rather than complexity of simulated scenario. More studies are needed to confirm this trend and to clarify the role of simulated stress response in a long-term learning scenario.