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Background Few data exist to guide the physical design of biocontainment units, particularly the doffing area. This can impact the contamination risk of healthcare workers (HCWs) during doffing of personal protective equipment (PPE). Methods In phase I of our study, we analyzed simulations of a standard patient care task with 56 trained HCWs focusing on doffing of high-level PPE. In phase II, using a rapid cycle improvement approach, we tested different balance aids and redesigned doffing area layouts with 38 students. In phase III, we tested 1 redesigned layout with an additional 10 trained HCWs. We assessed the effectiveness of design changes on improving the HCW performance (measured by occurrence and number of risky behaviors) and reducing the physical and cognitive load by comparing the results from phase I and phase III. Results The physical load was highest when participants were removing their shoe covers without any balance aid; the use of a chair required the lowest physical effort, followed by horizontal and vertical grab bars. In the revised design (phase III), the overall performance of participants improved. There was a significant decrease in the number of HCW risky behaviors (P =.004); 5 risky behaviors were eliminated and 2 others increased. There was a significant decrease in physical load when removing disposable shoe covers (P =.04), and participants reported a similar workload in the redesigned doffing layout (P =.43). Conclusions Through optimizing the design and layout of the doffing space, we reduced risky behaviors of HCWs during doffing of high-level PPE. [ABSTRACT FROM AUTHOR]