The heel pad consists of a superficial microchamber layer and a deep macrochamber layer. This study highlights the different biomechanical behaviors between the microchamber and macrochamber layers using ultrasonography. The heel-pad in each left foot of six healthy volunteers aged about 25 years old were measured with a device consisting of a 10-MHz linear-array ultrasound transducer and a load cell. The testing heels were loaded on the ultrasound transducer with a loading velocity of approximately 0.5 cm/s and were withdrawn when the specified maximum stress (158 kPa) was reached. Unloaded tissue thickness, end-loaded thickness, deformation proportion, average deformation and rebound rates and elastic modulus of the microchamber and macrochamber layers were assessed. The unloaded thickness of the microchamber layer was approximately 30 % of the macrochamber layer. The microchamber layer also had significantly less unloaded thickness, end-loaded thickness, mean deformation rate, mean rebound rate and deformation proportion than the macrochamber layer. A significant difference between the unloaded and end-loaded thickness in the macrochamber layer was observed. The average soft tissue deformation rate was significantly different from the rebound rate in the microchamber layer. A similar trend was detected in the macrochamber layer. The elastic modulus of the microchamber layer was 450±240 kPa, which was nearly 10 times of that in the macrochamber layer. In conclusion, ultrasound can identify the heterogeneous tissue properties of the heel pad. The macrochamber layer responds to loading with large deformation and the microchamber layer has a high degree of tissue stiffness.