Background: The use of N95 masks by Healthcare Providers (HCPs) has increased throughout the COVID-19 pandemic. Thus, the question arises as to whether wearing this type of mask influences the concentration of carbon dioxide (CO2) and oxygen (O2) in the blood. Methods: We analyzed CO2 and O2 concentrations, measured in millimeter mercury. Our inclusion criteria were as follows: wearing N95 mask continuously for four hours, and working in an acute care setting that mandates continuous physical activity. Excluded were those who interrupted the four hours and those who voluntarily quit the study. We measured the partial pressure of CO2 and O2 from venous blood samples taken before and after wearing the N95 mask. Results: The total number of participants included in our analysis was 43, with a 100% follow-up rate. We noted that there is a significant difference in pCO2 level between the first reading (M=42.37, SD=6.77) and the second reading (M=44.56, SD=6.39); t(42)= -201, p=0.05. We also noted a significant difference in the level of the first pO2 (M=55, SD=24.59) and the second pO2 readings (M=45.71, SD=24.46); t(42)=2.62, p<0.05. Conclusion: The wearing of the N95 mask for as long as four continuous hours is associated with the risk of hypoxia and a slight increase in CO2 concentrationsy.