The water vapor transmission rate (WVTR) of a fabric plays a pivotal role in managing next-to-the-skin moisture, which is essential for maintaining human thermophysiological comfort. Studies have reported an effect of the thickness of the air gap between human skin and the cloth layer, called the microclimate, but an understanding of the mechanisms involved is still missing. As a result, this study aims to investigate the effect of the microclimate thickness on the WVTR of a series of sportswear fabrics using the upright cup method following ASTM E96, Standard Test Methods for Gravimetric Determination of Water Vapor Transmission Rate of Materials. The WVTR of the fabrics initially decreased, then reached a minimum at a certain microclimate thickness. Above this critical air gap, the fabrics’ WVTR slightly increased and then reached a plateau at higher values of the air gap. The variation in the fabrics’ WVTR with the microclimate thickness was attributed to the change in the moisture vapor transport mechanism within the microclimate. Numerical simulations of the WVTR test using COMSOL Multiphysics showed that the moisture vapor transport mechanism shifted from diffusion-driven below the critical air gap to convection-driven flow above, which explains the increase in the WVTR above the critical air gap. The plateau in the fabrics’ WVTR at higher microclimate thicknesses was possibly attributed to the fact that the higher rotation speed of the convection cells compensated for the lower water evaporation rate in the cup. The fabrics’ WVTR exhibited a positive nonlinear relationship with the fabric’s air permeability. These findings will help improve fabric selection and garment design to enhance their thermophysiological comfort. Another major finding of this study is the need to better specify the value of the air gap in the upright cup method, to improve the reliability of fabrics’ WVTR measurement.