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Importance: The COVID-19 pandemic resulted in a widespread acute shortage of N95 respirators, prompting the Centers for Disease Control and Prevention to develop guidelines for extended use and limited reuse of N95s for health care workers (HCWs). While HCWs followed these guidelines to conserve N95s, evidence from clinical settings regarding the safety of reuse and extended use is limited., Objective: To measure the incidence of fit test failure during N95 reuse and compare the incidence between N95 types., Design, Setting, and Participants: This prospective cohort study, conducted from April 2, 2021, to July 15, 2022, at 6 US emergency departments (EDs), included HCWs who practiced N95 reuse for more than half of their clinical shift. Those who were unwilling to wear an N95 for most of their shift, repeatedly failed baseline fit testing, were pregnant, or had facial hair or jewelry that interfered with the N95 face seal were excluded., Exposures: Wearing the same N95 for more than half of each clinical shift and for up to 5 consecutive shifts. Participants chose an N95 model available at their institution; models were categorized into 3 types: dome (3M 1860R, 1860S, and 8210), trifold (3M 1870+ and 9205+), and duckbill (Halyard 46727, 46767, and 46827). Participants underwent 2 rounds of testing using a different mask of the same type for each round., Main Outcomes and Measures: The primary outcome was Occupational Safety and Health Administration-approved qualitative fit test failure. Trained coordinators conducted fit tests after clinical shifts and recorded pass or fail based on participants tasting a bitter solution., Results: A total of 412 HCWs and 824 N95s were fit tested at baseline; 21 N95s (2.5%) were withdrawn. Participants' median age was 34.5 years (IQR, 29.5-41.8 years); 252 (61.2%) were female, and 205 (49.8%) were physicians. The overall cumulative incidence of fit failure after 1 shift was 38.7% (95% CI, 35.4%-42.1%), which differed by N95 type: dome, 25.8% (95% CI, 21.2%-30.6%); duckbill, 28.3% (95% CI, 22.2%-34.7%); and trifold, 61.3% (95% CI, 55.3%-67.3%). The risk of fit failure was significantly higher for trifold than dome N95s (adjusted hazard ratio, 1.75; 95% CI, 1.46-2.10)., Conclusions and Relevance: In this cohort study of ED HCWs practicing N95 reuse, fit failure occurred in 38.7% of masks after 1 shift. Trifold N95s had higher incidence of fit failure compared with dome N95s. These results may inform pandemic preparedness, specifically policies related to N95 selection and reuse practices.