Air layers in multi-layer firefighter clothing ensembles resist heat transfer from the body to the environment. By reducing the volume of air between clothing layers, heat loss may be improved throughout the multi-layer firefighter turnout suit clothing system, potentially leading to reduced heat strain for the wearer. This research utilized a systems-level approach to the methodology in order to measure the effects of fabric properties and garment air gap dimensions on clothing system heat loss through specially configured turnout suit constructions. One experimental configuration incorporated a tight fitting stretchable moisture barrier garment. Another construction used thermal knit underwear to represent a closer fitting thermal liner. Air gap surface area, volume, and thickness were estimated using three-dimensional body scanning. This study showed the significant impact of fabric air permeability and clothing air gap volume on heat loss through structural firefighter suits. Tested individually, the tighter fitting moisture barrier construction permitted greater heat loss in comparison to the traditional fit moisture barrier. Heat loss differences associated with moisture barrier fit were not observed when the moisture barriers were configured in the three-layer turnout clothing system. This research showed that microclimate air gap volume is strongly correlated with total heat loss. It confirmed the significant impact of clothing air layers on heat loss through firefighter turnout systems.