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This study aimed to investigate the effects of two functional sport shirts with different fiber compositions (polyester: 100% polyester vs polyester-lyocell combination: 67%-33%) on thermoregulation and wearing comfort during treadmill walking at an average intensity under moderate environmental conditions. It was hypothesized that the hydrophilic lyocell fibers would benefit sweat evaporation and, thus, provide a superior cooling effect. Using a repeated-measures study design, core, surface and between skin-and-shirt temperatures were measured in 11 men while wearing either the polyester or polyester-lyocell shirt. Additionally, sweat loss, heart rate and subjective perception of comfort were determined. In spite of significantly greater sweat production, relative humidity was lower with the polyester-lyocell shirt. In the later phases of the walking intervention, the temperatures were consistently higher with polyester-lyocell, although between-shirt differences failed to reach significance. These results suggest that greater water retention limits the thermoregulatory efficiency of cellulosic fibers when excessive sweat production occurs.