Background: Healthcare workers (HCWs) are at increased risk of exposure to severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). Personal protective equipment (PPE) is mandated for HCWs. However, the physiological effects on the HCWs while working in the protective gear remains unexplored. This study aimed to assess the physiological effects of the prolonged use of PPE on HCWs. Materials and methods: Seventy-five HCWs, aged 18–50 years were enrolled in this prospective, observational, cohort study. The physiological variables [heart rate, oxygen saturation, and perfusion index (PI)] were recorded at the start of duty, 4 hours after wearing N95 filtering facepiece respirator (FFR), pre-donning, and post-doffing. The rating of perceived exertion (RPE) score and modified Borg scale for dyspnea was evaluated. The physiological variables were represented as the mean ± standard deviation. Wilcoxon signed-rank test was used to show any difference in RPE and modified Borg scale for dyspnea. A p value of <0.05 was considered significant. Results: There is a statistically significant difference in the physiological parameters post-doffing compared with baseline: Heart rate (p < 0.001); oxygen saturation (p < 0.001); PI (p < 0.001). RPE score showed increased discomfort with continuous use of N95 FFR. However, exertion increased only marginally. The major adverse effects noted with PPE use were fogging, headache, tiredness, difficulty in breathing, and mask soakage, with a resultant mean duration of donning to be 3.1 hours. Conclusion: The use of PPE can result in considerable changes in the physiological variables of healthy HCWs. The side effects may lead to excessive exhaustion and increased tiredness after prolonged shifts in the intensive care unit (ICU) while wearing PPE.