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The use of personal protective equipment (PPE) is required for the self-protection of healthcare workers during cardiopulmonary resuscitation (CPR) in patients at risk of aerosol transmission of infectious agents. The aim of this study was to analyze the impact of personal protective equipment on physiological parameters during CPR. A randomized, quasi-experimental, crossover design was used. The study was carried out in a training and simulation emergency box and the total sample consisted of 20 healthcare professionals. Two CPR tests were compared with the recommended sequence of 30 chest compressions and 2 ventilations. The duration of each test was 20 min. One of the CPR tests was carried out without using any PPE (CPR_control), i.e., performed with the usual clothing of each rescuer. The other test was carried out using a CPR test with PPE (i.e., CPR_PPE). The main variables of interest were: CPR quality, compressions, ventilations, maximum heart rate, body fluid loss, body temperature, perceived exertion index, comfort, thermal sensation and sweating. The quality of the CPR was similar in both tests. The maximum heart rate was higher in the active intervals (compressions + bag-valve-mask) of the test with PPE. CPR_PPE meant an increase in the perceived effort, temperature at the start of the thermal sensation test, thermal comfort and sweating, as opposed to CPR performed with usual clothing. Performing prolonged resuscitation with PPE did not influence CPR quality, but caused significant physiological demands. Rescuers were more fatigued, sweated more and their thermal comfort was worse. These results suggest that physical preparation should be taken into account when using PPE and protocols for physiological recovery after use should also be established.