Background: Undesirable side effects from wearing face masks during the ongoing COVID-19 pandemic continue to be discussed and pose a challenge to occupational health and safety when recommending safe application. Only few studies examined the effects of continuously wearing a face mask for more than one hour. Therefore, the influence of wearing a medical mask (MedMask) and a filtering facepiece class II respirator (FFP2) on the physiological and subjective outcomes in the course of 130 min of manual work was exploratively investigated. Physical work load and cardiorespiratory fitness levels were additionally considered as moderating factors. Methods: Twenty-four healthy subjects (12 females) from three different cardiorespiratory fitness levels each performed 130 min of simulated manual work with light and medium physical workload using either no mask, a MedMask or FFP2. Heart rate, transcutaneous oxygen and carbon dioxide partial pressure (PtcO2, PtcCO2) as well as perceived physical exertion and respiratory effort were assessed continuously at discrete time intervals. Wearing comfort of the masks were additionally rated after the working period. Results: There was no difference in time-dependent changes of physiological outcomes when using either a MedMask or a FFP2 compared to not wearing a mask. A stronger increase over time in perceived respiratory effort occurred when the face masks were worn, being more prominent for FFP2. Physical workload level and cardiorespiratory fitness level were no moderating factors and higher wearing comfort was rated for the MedMask. Conclusion: Our results suggest that using face masks during light and medium physical manual work does not induce detrimental side effects. Prolonged wearing episodes appeared to increase respiratory effort, but without affecting human physiology in a clinically relevant way.