OBJECTIVE: To assess the physiological impact of the N95 filtering facepiece respirator (FFR) on healthcare workers. METHODS: Ten healthcare workers each conducted multiple 1-hour treadmill walking sessions, at 1.7 miles/h, and at 2.5 miles/h, while wearing FFR with exhalation valve, FFR without exhalation valve, and without FFR (control session). We monitored heart rate, respiratory rate, tidal volume, minute volume, blood oxygen saturation, and transcutaneously measured PCO2. We also measured user comfort and exertion, FFR moisture retention, and the carbon dioxide and oxygen concentrations in the FFR's dead space. RESULTS: There were no significant differences between FFR and control in the physiological variables, exertion scores, or comfort scores. There was no significant difference in moisture retention between FFR with and without exhalation valve. Two subjects had peak PCO2 ≥ 50 mm Hg. The FFR with exhalation valve offered no benefit in physiological burden over the FFR without valve. The FFR dead-space oxygen and carbon dioxide levels did not meet the Occupational Safety and Health Administration's ambient workplace standards. CONCLUSIONS: In healthy healthcare workers, FFR did not impose any important physiological burden during 1 hour of use, at realistic clinical work rates, but the FFR dead-space carbon dioxide and oxygen levels were significantly above and below, respectively, the ambient workplace standards, and elevated PCO2 is a possibility. Exhalation valve did not significantly ameliorate the FFR's PCO2 impact.