Thermal comfort is an important criterion for the overall wear comfort of shoes. In this study, the materials properties, the microclimate of two types of sport shoes made of leather and mesh spacer fabric were evaluated. The human subjective sensations when wearing the shoes were investigated. Ten youth and ten elderly subjects have taken part in wear trials that consist of three activities: sitting, walking and running, while wearing the two types of shoes and in the barefoot condition. The order of the wear trials is randomized for each subject. The temperature and humidity at six locations of the foot were recorded during the wear trials and subjective perceptions of thermal, humidity and comfort were rated at the end of each wear trial. The results show that the leather sports shoes trap more heat and moisture on the feet than the mesh fabric sports shoes. The effect is more significant during running. Therefore, the shoe upper materials not only affect the temperature and humidity of the foot dorsal but also those of the foot plantar. Gender, foot condition and type of activity have a significant linear relationship with thermal, humidity and comfort perceptions towards footwear. Age does not have any impact on the three subjective perceptions. The findings in this study can therefore act as a reference source for the design and development of footwear that have better wear comfort.