In this study, three knitted fabrics were developed for a single garment: plain knitted polyester (Polyethylene terephthalate, 120 D), plain knitted polyester with a plaited structure (120 D), and plain knitted polyester with a plaited structure (120 D and 20/80 C/P yarns). The plain polyester fabric was treated with cutinase enzyme. The effects of enzymatic treatment and knit structure on thermo-physiological comfort were evaluated. The cutinase treatment has not revealed a significant change on the polyester fabrics. However, the knit structure results showed that the plaited fabric with 120 D polyester and 20/80 C/P yarns exhibited the highest air permeability (264 cm 3 /cm 2 /s), thermal conductivity, and wickability, along with the lowest thermal resistance, due to its longer loop length (3.3 mm). Based on fabric properties and wearer physiology, a single sportswear T-shirt was designed: plain fabric for the chest and hips, plaited fabric with fewer knit/miss/tuck stitches for the shoulders and abdomen, and plaited fabric with more knit/miss/tuck stitches for the upper and central back. The study demonstrates that a single garment combining these structures can achieve optimal thermo-physiological comfort for sportswear.