The scientific literature highlights how Mixed Reality (MR) simulations allow obtaining several benefits in healthcare education. Simulation-based training, boosted by MR, offers an exciting and immersive learning experience that helps health professionals to acquire knowledge and skills, without exposing patients to unnecessary risks. High engagement, informational overload, and unfamiliarity with virtual elements could expose students to cognitive overload and acute stress. The implementation of effective simulation design strategies able to preserve the psychological safety of learners and the investigation of the impacts and effects of simulations are two open challenges to be faced. In this context, the present study proposes a method to design a medical simulation and evaluate its effectiveness, with the final aim to achieve the learning outcomes and do not compromise the students' psychological safety. The method has been applied in the design and development of an MR application to simulate the rachicentesis procedure for diagnostic purposes in adults. The MR application has been tested by involving twenty students of the 6th year of Medicine and Surgery of Università Politecnica delle Marche. Multiple measurement techniques such as self-report, physiological indices, and observer ratings of performance, cognitive and emotional states of learners have been implemented to improve the rigour of the study. Also, a user-experience analysis has been accomplished to discriminate between two different devices: Vox Gear Plus® and Microsoft Hololens®. To compare the results with a reference, students performed the simulation also without using the MR application. The use of MR resulted in increased stress measured by physiological parameters without a high increase in perceived workload. It satisfies the objective to enhance the realism of the simulation without generating cognitive overload, which favours productive learning. The user experience (UX) has found greater benefits in involvement, immersion, and realism; however, it has emphasized the technological limitations of devices such as obstruction, loss of depth (Vox Gear Plus), and narrow FOV (Microsoft Hololens).