Background: Face masks have become an important part of the COVID-19 prevention approach. This study aimed to explore the effect of wearing masks on exercise ability and ventilatory anaerobic threshold (VAT). Material/Methods: Thirty-four young, healthy volunteers were included in this study, consisting of 18 men and 16 women. The subjects were randomized to perform 2 cardiopulmonary exercise tests (CPET) on a cycle ergometer with gas exchange analysis, one with and another without wearing a face mask (cross-over design). The general data for all subjects and indicators from the 2 exercise tests performed with and without wearing a face mask were collected. Results: In cardiopulmonary exercise tests, wearing a mask significantly (P<0.05) decreased peak indexes (eg, work rate (WR), oxygen consumption per kg body weight (VO2/kg), heart rate (HR), ventilation per minute (VE) and carbon dioxide ventilation equivalent (VENCO2)) and anaerobic threshold indexes (eg, WR, HR, VE, breath frequency (BF), dead space ratio (VD/VT), and VENCO2). However, the PETCO2 at peak was significantly higher. There was a positive linear correlation between WR difference and VO2 difference at VAT (abbreviated as delta WR@ VAT and delta VO2@VAT, respectively) (r=0.495, P=0.003). Subgroup analysis of the VAT indexes showed that WR, VO2/kg, and VE were significantly decreased in the advanced VAT group with mask compared with the stable VAT group with mask (P<0.05). Logistic regression showed that delta VE, delta BF, and delta VE/VCO2 had independent influences on VAT. Conclusions: Wearing masks advances VAT in healthy young subjects during CPET. The advanced VAT was associated with changes in VE, BF, and VE/CO2 while wearing masks.