Objectives: The study investigated whether nurses' different working schedules are associated with different levels of job-relatd strain, health symptoms and behavior. No reports have been accessible in the relevant literature on the possible association between shift work and job-related strain in nurses. Materials and Methods: This was a cross-sectional study conducted at a large university hospital in North-East Italy, involving 806 nurses working in selected departments. A multilevel logistic regression was applied to assess the association between work shift conditions and selected outcomes. Results: Night shifts were associated not only with higher odds of having a high Job Demand, but also with lower odds of having a high Decision Authority and consequently with a stronger likelihood of having higher levels of Job Strain (high Job Demand score≥ 38 and Low Decision Authority). The night shift was associated with various symptoms, particularly exhaustion (p = 0.039) and gastric pain (p = 0.020). Nurses' working schedules did not affect their job satisfaction scores. Conclusions: It has been confirmed that night shifts are a risk factor for nurses' health perception and working night shifts carries a considerable degree of strain. This is a condition that hospital nursing managements need to consider carefully to avoid burnout in nursing personnel and prevent an excessive turnover in this profession, which is a recurring problem for health care organizations.