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Background: Operating rooms have stressful and complex working environments. Working in a stressful environment and exposure to various risk factors could affect human health both physiologically and psychologically and causes fatigue. The purpose of this study was to examine fatigue levels and factors affecting fatigue in operating room nurses. Methods: This research was a descriptive, cross-sectional study. The study sample comprised 99 nurses working in operating rooms of hospitals in a city in Aegean Region of Turkey. Data were gathered with a descriptive characteristics form and the Checklist Individual Strength between December 2019 and February 2020. Data analysis was performed by using numbers, percentages, mean, standard deviation, Student's t-test, one-way ANOVA, Kruskal-Wallis, Mann-Whitney-U and Pearson correlation tests. The STrengthening the Reporting of OBservational studies in Epidemiology (STROBE) checklist for cross-sectional studies was used for reporting study data. Results: The mean age of the nurses was 30.68±8.78 years, the mean duration of operating room experience was 5.37±4.78 years and the mean working time was 48.65±5.93 h/week. The lowest and the highest fatigue scores of the nurses were 34 and 128 respectively with a mean of 77.53±18.76. A significant difference was found between fatigue levels according to presence of a chronic disease, type of hospital, activity levels, job titles and types of work schedules (p |