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Thermo-physiological comfort of automotive seating is governed by the complex interaction between seat-cover materials, their structural configuration, and the heat and moisture exchange occurring at the seat–body interface during prolonged sitting. While numerous studies have examined individual textile constructions or isolated comfort parameters, integrated evaluations combining objective material testing with dynamic microclimate measurements under realistic loading conditions remain limited. This study thoroughly examined six commercially important vehicle seat-cover materials that represent laminated, warp-knitted, and woven polyester architectures. Standardized laboratory techniques were used to quantify objective comfort qualities, such as air permeability, water vapor permeability, thermal resistance (Rct), and evaporative resistance (Ret) and transient heat flux test (H-test). Simultaneously, a multi-sensor system was used to constantly monitor temperature and relative humidity at the seat–body interface during sitting loading in a controlled subjective microclimate experiment at room temperature. The findings show that lamination technique and textile structure have a major impact on both transient microclimate behavior and steady-state material properties. Increased air and moisture transmission in warp-knitted and more open structures resulted in reduced evaporative resistance and more stable microclimate conditions. Denser laminated structures, on the other hand, exhibited more resistance to heat and evaporation, which led to a greater buildup of moisture when they were seated. Different temporal responses in temperature and humidity were also shown by the multi-sensor microclimate studies, underscoring the significance of assessing comfort beyond static material metrics. This study demonstrates that static thermos-physiological parameters alone are not sufficient to predict real stated comfort behavior. By integrating time-resolved microclimate analysis under realistic seated loading with standardized testing, a more reliable evaluation framework for automotive seat-cover comfort is proposed. Full article