Background: Clinical simulation is a valuable tool used in healthcare education to develop technical and cognitive skills in a controlled, low risk setting. Despite their prevalence, the extent to which simulations replicate the physiological stress responses of real clinical scenarios remains underexplored. This study aimed to evaluate the relationship between self-reported baseline anxiety, perceived taskload, and physiological stress responses among healthcare professionals and students during a simulation patient monitoring intervention. Methods: Twenty-two participants, including licensed nurses, medical assistants, and clinical students, monitored a patient simulator as it progressed through a programmed scenario with deteriorating clinical states. Participants were fitted with biometric monitoring sensors to measure heart rate, blood pressure, stress, and facial expressions. Results: Galvanic Skin Response (GSR) data indicated participants experienced a significant increase in stress during the patient’s extreme deterioration phase compared to baseline. Two GSR measurements were analyzed: peaks per minute (p-value = 0.002) and average peak amplitude (microsiemens, p-value < 0.001). Conclusion: A simulated clinical scenario can elicit significant increases in psychological stress, even in controlled and low risk environments. These findings support the utility of simulation both as a training tool and as a research methodology for assessing the cognitive and emotional demands experienced by clinicians in high-stress environments.