Introduction: as the largest workforce in the healthcare industry, nurses are essential to maintaining patient safety. Personal and environmental factors, including high demands, irregular work hours, and large workloads, make nurses vulnerable to fatigue. Method: the study involved 60 participants using several tools for data collection, including the NASA Task Load Index (NASATLX) to assess workload, the Swedish Occupational Fatigue Inventory (SOFI) to measured fatigue, heart rate variability (HRV) measurements to evaluated autonomic nervous system activity, electroencephalogram (EEG) analyzed for cognitive functions, and the Psychomotor Vigilance Test (PVT) to assessed cognitive performance. Results: the findings revealed that the nurses experienced high subjective workloads, particularly related to mental, physical, and temporal demands. Fatigue, especially in terms of energy depletion and sleepiness, was significantly reported. HRV data indicated a shift toward parasympathetic dominance after each shift, while EEG results showed decreased theta and alpha wave activity, suggesting increased fatigue. The PVT results showed slower reaction times and more lapses in performance, especially after night shifts, indicating cognitive impairment due to fatigue. Conclusions: these results highlight the considerable impact of high workloads, shift work, and fatigue on the health and performance of nurses. The study suggests that healthcare institutions should implement strategies to reduce workload, manage fatigue, and improve recovery to maintain optimal cognitive performance and overall wellbeing of nursing staff. These findings can inform policies aimed at improving working conditions and ensuring better care delivery in hospital settings. © 2025; Los autores.