This study investigates the intricate relationship between moisture and thermophysiological comfort in multilayered fabric assemblies, particularly focusing on the impact of simulated sweat in comparison to distilled water. The multilayered fabric assembly under investigation consists of an outside layer of breathable nylon coated in polyurethane (PU), an inner layer of polyester knit fabric, and an intermediate layer with micro-polyester wadding, hollow-polyester wadding, or spacer fabric. The findings show that distilled water has a greater impact on thermal conductivity than simulated sweat, and this influence is considerable on the thermal characteristics of materials when they are wet. Additionally, the research emphasizes the substantial role of fabric construction and thickness in moisture behavior. Analyzing various layered ensembles, it is observed that polyester spacer fabric as the middle layer consistently outperforms hollow and micro-polyester waddings in terms of water vapor permeability, regardless of the moisture type (water or sweat). Also, it is found that heavier and thicker fabrics exhibit lower moisture vapor permeability. The study underscores the need for realistic sweat conditions in textile performance assessments.