Simulation is regarded as an effective educational method for the delivery of clinical scenarios. However, exposure to unfamiliar environments during simulation can cause excessive stress among students, possibly leading to unnatural speech/behavior and poor skill learning (Yerkes-Dodson’s law). Thus, assessing students’ stress in a simulation can provide educators with a better understanding of their mental state. This study sought to clarify stress changes throughout the progression of the simulation by measuring heart rate variability and students’ subjective reactions in 74 nursing students. Heart rate variability was calculated in terms of its high-frequency (HF) and low-frequency/high-frequency (LF/HF) components during 4 phases—the break, patient care, reporting, and debriefing. Students were interviewed about stress experienced during the simulation. The results showed that HF decreased significantly from the break to the patient care and reporting phases. Furthermore, LF/HF increased significantly from the break to the reporting phases. Approximately 55 students felt stressed during the simulation, 24 of whom felt most stressed during the reporting phase. Therefore, the reporting phase involved high objective and subjective stress. It may be possible that the educator’s evaluative attitude increased students’ stress. Therefore, a stress intervention during the reporting phase might further improve students’ performance during that phase. The debriefing phase did not significantly differ from the break phase for objective stress, and students did not report feeling stressed. Thus, in this phase, they were released from the stress of the reporting phase and the unfamiliar environment. During this phase, they might be able to learn what they could not understand owing to high stress in the patient care and reporting phases. This study provides objective and subjective evidence of students’ stress during simulation, and indicates the necessity of providing support during the reporting phase and the importance of debriefing when using clinical scenarios for teaching clinical skills.