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This article presents a research methodology for analysis of stress effects and allostatic load of nurses during daily activities. Stress-related health issues are critical in healthcare workers, in particular nurses. Typical causes of stress include inadequate staffing of nurses for the number and acuity of patients, dealing with difficult patients and families, and lack of autonomy in care delivery decisions. This is all compounded by lack of recovery time while on shift, variable shifts with limited recovery time between days worked, and fatigue from dealing with difficult patients, families, and healthcare workers. Under unresolved stress, the heart rate and other vital parameters may fail to return to the baseline. This study examined the physiological responses of nurses during care on a high-fidelity patient simulation to develop a research methodology and identify physiological parameters suitable for real-time assessment of allostatic load during work. Our results demonstrated that heart rate and heart rate variability can be reliably measured using wearable sensors to assess allostatic load. During this study and our previous related work, we acquired valuable experience regarding selection and deployment of commercially available sensors, system integration, recruitment of subjects, and general research methodology. The research methodology developed and presented in this article can be applied to a number of other applications and experimental protocols.