Studying the in-shoe microclimate of older individuals is important for enhancing their foot comfort and preventing foot diseases. However, there is a lack of scientific work that explores the thermo-physiological wear comfort of older individuals with different footwear. This study aims to examine the effects of upper footwear materials on changes and distributions in the foot skin temperature and relative humidity for older individuals. Forty older individuals are recruited to perform sitting and walking activities under four experimental conditions in a conditioning chamber. The findings indicate that footwear upper constructed of highly permeable mesh fabric with large air holes shows fewer changes in foot skin temperature (ranging from 1.3 to 3.3 °C) and relative humidity (ranging from −13.3 to 5.7%) throughout the entire foot during dynamic walking, as well as higher subjective ratings on perceived thermal comfort when compared to footwear made of synthetic leather and composite layers. The findings serve to enhance current understanding of designing footwear with optimum comfort for older adults.