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Han Thi Ngoc Le,1,* Kien Xuan Nguyen,2,* Toan Duy Nguyen,1 Ho Quang La,3 Xuan Thanh Nguyen,4 Dung Tien Le,5 Huy Quoc Pham,5 Khai Xuan Nguyen,6 Hiep Hoang Nguyen,7 Viet Van Pham,6 Cong Van Pham,5 Dinh Cong Pho,8 Binh Nhu Do,8 Tien Viet Tran,9 Son Tien Nguyen,10 Tuan Dinh Le,10,* Thuc Cong Luong1 1Cardiovascular Center, Military Hospital 103, Vietnam Military Medical University, Hanoi, 10000, Vietnam; 2Department of Military Medical Command and Organization, Vietnam Medical Military University, Hanoi, 10000, Vietnam; 3Hospital Quality Control Division, Military Hospital 103, Vietnam Military Medical University, Hanoi, 10000, Vietnam; 4Senior Officer Department, Military Hospital 103, Vietnam Military Medical University, Hanoi, 10000, Vietnam; 5Intensive Care, Emergency and Poison Control Center, Military Hospital 103, Vietnam Military Medical University, Hanoi, 10000, Vietnam; 6Interventional Radiology Department, Military Hospital 103, Vietnam Military Medical University, Hanoi, 10000, Vietnam; 7Field Internal Medicine Center, Military Hospital 103, Vietnam Military Medical University, Hanoi, 10000, Vietnam; 8Department of Military Science, Vietnam Military Medical University, Hanoi, 10000, Vietnam; 9Department of Infectious Diseases, Military Hospital 103, Vietnam Medical Military University, Hanoi, 10000, Vietnam; 10Department of Rheumatology and Endocrinology, Vietnam Military Hospital 103, Medical Military University, Hanoi, 10000, Vietnam*These authors contributed equally to this workCorrespondence: Thuc Cong Luong, Cardiovascular Center, Military Hospital 103, Vietnam Military Medical University, 160 Phung Hung Street, Phuc La Ward, Ha Dong District, Hanoi, Vietnam, Email luongcongthuc@vmmu.edu.vnBackground: Personal protective equipment (PPE), an essential shield to protect healthcare workers (HCWs) during the COVID-19 pandemic, has been reported to affect their heart rate variability (HRV).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.Keywords: cardiovascular disorders, cardiovascular change, heart rate variability, personal protective equipment, healthcare workers, COVID-19