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Objective: To investigate the changes of very short-term heart rate variability in HCWs after three hours of wearing PPE to treat COVID-19 patients at different working times and intensities, and related factors. Methods: Sixty-five healthy HCWs were enrolled at the Number 2 Infectious Field Hospital (formed by Military Hospital 103), Vietnam. Two-minute 12-lead electrocardiograms were recorded before wearing and after removing PPE. Results: After three hours of wearing PPE, the mean heart rate of HCWs increased (p = 0.048) meanwhile, the oxygen saturation decreased significantly (p = 0.035). Standard deviation of all normal to normal intervals (SDNN), mean intervals RR (mean NN), and root mean square successive difference (rMSSD) after wearing PPE was also reduced significantly. SDNN, Mean NN, and rMSSD decreased as the working intensity increased (as in mild, moderate, and severe patient departments). In univariate regression analysis, logSDNN, logmean NN and logrMSSD were positively correlated with SpO2 and QT interval (r = 0.14, r = 0.31, r = 0.25; r = 0.39, r = 0.77, r = 0.73, respectively) and were negatively correlated with ambient temperature inside PPE (r = − 0.41, r = − 0.405, r = − 0.25, respectively) while logmean NN and log rMSSD were negatively correlated with diastolic blood pressure (r = − 0.43, r = − 0.39, respectively). In multivariable regression analysis, logSDNN and logmean NN were negatively correlated to ambient temperature inside PPE (r = − 0.34, r = − 0.18, respectively). Conclusion: Time-domain heart rate variability decreased after wearing PPE. Time-domain HRV parameters were related to ambient temperature inside PPE, diastolic blood pressure, QT interval, and SpO2. [ABSTRACT FROM AUTHOR]