The effect of thermal properties on the three-dimensional knitted spacer fabrics made from functional fibers (i.e. Outlast (R), Coolmax (R)) with different fiber compositions was studied. The spacer fabrics were specifically designed for mattress ticking applications. Samples were manufactured with two fabric tightness and knit designs, and four Outlast (R) fiber compositions. Thermal conductivity, thermal resistance, thermal absorptivity, thermal diffusivity, and relative water vapor permeability were considered as thermal comfort properties. Alambeta and Permetest devices were used for the measurement of thermal properties. Fabric design was the leading criteria on the thermal resistance and water vapor permeability, while fiber compositions became more important on the thermal absorptivity. The contribution of Outlast (R) fiber on the thermoregulatory efficiency of spacer fabrics was analyzed using a differential scanning colorimeter. The thermoregulatory effect of Outlast (R) fiber was slightly observed in the 33% Outlast (R) fiber composition. Water vapor permeability of open-skin samples was higher than the closed-skin samples, which was due to the holed/meshed structure of the open-skin structure for the same fiber content and fabric construction. Statistical analysis was also performed and confirmed the contribution of each factor, including their interactions. In particular, the interaction became more significant than the main factors for thermal diffusion behavior of samples.