Objective: Since the emergence of coronavirus disease, health care professionals in high-risk environments are mandated to wear N95 respirators for prolonged periods. The effect of this prolonged use on cardio-respiratory variables and gas-exchange is poorly defined. The objective of the current study was to determine gas exchange abnormalities and physiological changes among healthcare workers during a 4-hour emergency department (ED) shift while wearing the N95 respirator. Methods: This was a single-center prospective observational study that consisted of medical staff working at the Sheba Medical Center ED. Physiological effects and gas exchange variables were obtained under normal breathing conditions and after 4-hour shifts while continuously wearing an N95 respirator. Comparisons of paired measurements were performed using a non-parametric Wilcoxon matched-pairs signed-rank test. Results: Forty-one subjects were included in the study. Prolonged N95 respirator use was associated with a significant decline in plasma pH [7.35mmHg vs. 7.34mmHg, P=0.02], PvO2 [23.2 mmHg vs. 18.6 mmHg, P<0.001] and a concurrent increase in EtCO2 [32.5mmHg vs. 38.5mmHg, p<0.0001]. PvCO2 and bicarbonate levels did not differ. No significant change was observed for heart rate or oxygen saturation. Conclusion: Using an N95 respirator for prolonged periods by healthcare professionals may provoke changes in gas exchange. The clinical significance of these changes in terms of symptoms or longer-term health status is unknown and remains to be determined. Key Words: N95 Respirator, Physiological effects, SARS CoV 2.