This study investigated the relationship of clothing microclimate and physiological responses in order to examine the layering effects on the clothing microclimate as an index to predict clothing thermal insulation ($I_{cl}$). Experiments were conducted in a $15^{circ}C$ environment on six physically active males. Increased clothing layers resulted in higher mean temperature inside the clothing ($ar{T}_{cl}$) and $I_{cl}$. The $I_{cl}$ had a high correlation with: $ar{T}_{cl}$ (r = 0.556), the difference between the innermost surface temperature and the outermost surface temperature at the chest (DST) (r = 0.549) and the temperature inside clothing at the abdomen (r = 0.478). $ar{T}_{cl}$ had the highest correlation with the temperature inside clothing at the abdomen (r = 0.889). $ar{T}_{cl}$ also had the highest correlation with $ar{T}_{sk}$ (r = 0.860). The results showed that the relationship between $I_{cl}$ and $ar{T}_{cl}$ was linear (p < .01). Thermal comfort had a negative correlation with $ar{T}_{cl-thigh}$ (r=-0.411) and $ar{T}_{cl}$ (r = -0.323) (p < .01.)