The present study investigated the short-term effects of using Personal Protective Equipment (PPE) and physical workload intensity on human physiological and cognitive performance among 21 males and 19 females. PPE1 consisted of a medical coverall and surgical mask, while PPE2 consisted of impermeable full-body coverall, shoe covers, latex gloves, N95 mask, and face shield. Objective assessments were heart rate, energy expenditure, core body temperature, clothing microclimate temperature and humidity, and cognitive performance were the continuous performance test and N-Back test. Subjective assessments included thermal sensation, perceived fatigue/skin wetness/clothing moisture. Using PPE2 and increased workload intensity significantly increased the values of all physiological parameters and the subjective ratings of fatigue, thermal sensation, skin wetness, and clothing moisture. Moreover, the participants’ cognitive performance was not affected by the type of PPE. Practitioner summary: Healthcare workers are at the highest risk in the fight against pandemics. Therefore, these people are required to use personal protective equipment. Using this equipment may have difficulties. The results show physiological strain and higher subjective ratings associated with using full-body hospital PPE and increased physical workload.