Fatigue is highly prevalent among healthcare workers and is associated with impaired health, reduced productivity, and increased occupational risk. Fatigue assessment using standardized scales may provide a practical approach for identifying workers at risk of functional decline and guiding workplace health promotion strategies. However, its association with objectively measured physical fitness remains unclear in occupational healthcare settings.This study aimed to examine whether moderate-to-severe personal or work-related fatigue, assessed using validated fatigue scales, is independently associated with physical fitness performance, such as muscle strength, muscle endurance, flexibility, cardiorespiratory fitness, and grip strength among hospital employees.In this multicenter, cross-sectional study, personal and work-related fatigue were assessed using validated scales, and participants were categorized into mild or moderate-to-severe fatigue groups. Independent t-tests were conducted to compare demographic characteristics and physical fitness parameters between fatigue groups. Multivariate linear regression analyses were performed to examine associations between fatigue severity and physical fitness outcomes, particularly muscle strength and muscle endurance, adjusting for potential confounders.A total of 365 hospital employees were enrolled, with a mean age of approximately 45 years. Compared with employees reporting mild fatigue, those in the moderate-to-severe fatigue group were significantly younger (43.2 vs. 46.1 years, p < 0.05), exhibited poorer muscle strength and endurance as measured by sit-up normative scores (3.54 vs. 3.94, p < 0.05), and had lower overall physical fitness scores (11.8 vs. 12.7, p < 0.05). After multivariable adjustment, moderate-to-severe fatigue remained independently associated with lower muscle strength and endurance (β = −0.10; 95% CI: −0.19 to −0.01; p = 0.026). Compared with employees reporting mild fatigue, those with moderate-to-severe fatigue demonstrated an approximately 0.1-point reduction in muscle strength and endurance performance.Fatigue severity, assessed using standardized scales, was independently associated with poorer muscle strength and endurance, suggesting that fatigue may capture aspects of functional health status not fully reflected by traditional cardiometabolic or anthropometric indicators.Fatigue assessment may serve as a scalable and actionable tool for healthcare employers to identify workers at risk of functional decline and to guide targeted workplace health promotion and fatigue management strategies. Keywords (MeSH Terms) Fatigue; Physical Fitness; Workplace Health Promotion