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Abstract Background Simulation has been recognized as a shift in healthcare 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 interfere with students' abilities leading to unexpected emotional responses. The aim of this study is to determine if two simulation methods, high-fidelity (HF) and procedural simulation (PS), may be associated with stress responses at a self-perceived and biological level (salivary cortisol variations), and to compare stress levels of the two different simulation method. We also wanted to find independent variables associated with cortisol total hormonal output. Methods A quasi-experimental before-after study was used including the administration of questionnaires, and biomarkers evaluation by salivary cortisol samples before and after simulation. A total of 148 students 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 PT simulations. For NASA-TLX we performed a T-test mean comparison between groups. We used paired T-test mean comparison for cortisol analysis. Multivariable linear regression has been used to assess variables associated with AUCg values and perceived stress. Results values of STAI-Y scores were relatively higher at the end of the HF and PS sessions. NASA-TLX was significantly higher at baseline for the HF simulations, with respect to the PS simulation. Cortisol fold increase was significantly different in the two groups. Linear regression showed that cortisol AUCg was related to the STAI-Y score in both groups. Conclusion Participating students developed a stress response both after in the HF and PS training, testified by psychological and biological outputs. According to our results, stress levels were increased for simply being in a simulation scenario than to the intrinsic complexity of the task required. More studies are needed to confirm this trend and to clarify the role of simulated stress response in a long-term learning scenario.