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Background: Sleepiness and the changes in vital signs affect the quality of work of hospital staff. This study aimed to investigate the relationship between sleepiness and physiological changes and vital signs in night shift workers in rehabilitation staff. Materials and Methods: This study was a cross-sectional study performed on night shift personnel of Rafideh Hospital in Tehran City, Iran, in 2018. A questionnaire, including demographic information and the Stanford sleepiness scale, was used for data collection. The results were analyzed using repeated measures analysis in SPSS software v. 21. Results: The Mean±SD age of the participants was 38.23±5.96 years, comprising 33 men (64.7%) and 18 women (35.3%). Most personnel were married (72.5%) and non-smoker (82.4%). The amount of sleepiness increased significantly with increasing working time. Demographic characteristics had no significant association with sleepiness. However, there was a significant negative association between sleepiness and heart rate, and oral temperature. Blood pressure showed a decreasing trend with increasing working time until midnight. Also, an increase in blood pressure was recorded at 2 AM. Conclusion: The findings of this study showed that sleepiness might have a significant association with vital signs in night work personnel. Hence, proper planning of shift work schedules and compatibility with physiological conditions might result in better working conditions for medical staff. [ABSTRACT FROM AUTHOR]