Daily Sitting Time and Musculoskeletal Symptoms Are Associated with Poorer Health-Related Quality of Life Among University Employees

University employees are increasingly exposed to prolonged sitting, yet the relationships between daily sitting time, musculoskeletal symptoms (MSS), and health-related quality of life (HRQoL) in this occupational population remain insufficiently understood. This study aimed to estimate the prevalence, anatomical distribution, and chronicity of MSS among university employees and to explore their associations with daily sitting time and sociodemographic, occupational, and behavioural factors, as well as the relationships of these factors and MSS with HRQoL. A cross-sectional study was conducted among 893 academic and non-academic employees at a Spanish university. Participants completed an online questionnaire including sociodemographic and occupational characteristics, the Nordic Musculoskeletal Questionnaire, the 12-item Short Form Health Survey (SF-12), and the International Physical Activity Questionnaire. Multivariable logistic and linear regression models were used to examine factors associated with MSS and HRQoL. Female sex (OR = 1.58; 95% CI: 1.07–2.32), younger age (OR = 0.98; 95% CI: 0.96–0.99), and longer daily sitting time (OR = 1.001; 95% CI: 1.000–1.002) were associated with MSS after mutual adjustment. Overall, 82.8% of participants reported MSS during the previous 12 months, most frequently affecting the back (58.8%) and neck (49.2%). Daily sitting time was also associated with poorer mental HRQoL after mutual adjustment, whereas duration of work limitation due to MSS showed the strongest association with poorer physical HRQoL. Symptom prevalence was similar in academic and non-academic staff, although non-academic employees reported greater symptom chronicity and treatment use. MSS are highly prevalent among university employees and are associated with poorer HRQoL. Daily sitting time represents a potentially modifiable behavioural factor that should be considered in health promotion strategies within higher education institutions.