Purpose: The purpose of this study was to quantify the physical and psychological changes in staff health after moving from an open bay (OB) Neonatal Intensive Care Unit (NICU) to a single patient room (SPR) design. Subjects: 81 registered nurses, respiratory therapists, and neonatal nurse practitioners working in a 45 bed, regional referral, level IV NICU were recruited for the study. 59 participants completed all study activities. Design: A pre-post experimental design was used. Methods: The number of steps taken during a routine work shift were measured, for 3days, first in the OB NICU, and then in the SPR NICU, 3 months after the move. Blood pressure and body weight were measured in both locations as a measure of physical health. Participants also completed a questionnaire to assess chronic health conditions, musculoskeletal health, and involvement in weight control and exercise programs. Mental health was measured before and after the move with the Psychiatric Symptoms Index (PSI), and a brief survey about perception of the work environment, including space, light, and noise. Results: Participants walked, on average, nearly 900 steps more each day ( just over 1/3 of a mile) in the SPR NICU (6082.41) than the OB NICU (5185.23), p=0.014; physiologic parameters were unchanged. Hypertension was identified in 2 participants previously unaware of the condition; there were no changes in musculoskeletal complaints. Mental health was unchanged despite a significant increase in satisfaction with the amount of work and personal space (p<0.001), noise level (p<0.001), and natural light (p<0.001), in the new SPR NICU compared to the traditional OB NICU. NICU staff had high levels of anxiety and depression; 32% reported symptoms associated with clinical depression. Conclusions: New NICU designs and technological advances in monitoring and communication are thought to improve the quality of health care, family satisfaction and ease workflow burden for staff.There was a modest increase in the physical demands of the work environment in the SPR compared to OB NICU, which was neither beneficial nor harmful to staff’s physical health. Despite increased satisfaction with the physical space, psychological health was unchanged, with a high proportion of staff reporting symptoms associated with anxiety and depression. Although redesigned NICUs are appreciated for their aesthetics, the new work environment may not contribute to improvement in staff mental health.