The N95 filtering facepiece respirator (FR) is the most commonly recommended respiratory protection used in healthcare settings. However, concerns have been raised about its use because it can increase respiratory resistance and dead space. The primary objective of this study was to determine the effect of wearing N95 masks on the vital signs, i.e., oxygen saturation, pulse rate, and respiratory rate, of the participant health workers. Our secondary objective was to assess the subjective feeling of discomfort when wearing N95 masks. The study participants were healthy healthcare workers taking care of coronavirus disease 2019 (COVID-19)-infected pediatric cases who did at least six hours of continuous shift duty in the pediatric COVID-19 ward at a tertiary care hospital in the eastern part of India. They were evaluated for vital signs at various time intervals while wearing N95 masks. Subjective discomfort at any point in time was also noted. We found a significant variation in the mean oxygen saturation (SpO2) and heart rate (HR) reduction across the four different points. The pair-wise comparison showed a small but significant decrease in the mean SpO2 of 98.3% (1.1)at six hoursas compared with a mean SpO2 of 98.7% (0.9) at three hours. Similarly, a significant increase was noted for a mean HR of 84.7bpm (11.2) at six hours compared with a baseline of 82.3bpm (9.2) and 83.2bpm (8.8) at three hours. The continuous use of an N95 mask leads to a mild increase in respiratory rate. However, heart rate and oxygen saturation vary significantly at different points in time after N95 mask use.