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Objective: Prolonged wheelchair use following spinal cord injury is commonly associated with the development of pressure ulcers. This creates the need to better understand the anatomy of the buttocks. The aim was to assess the reliability of using ultrasound (US) to study buttocks soft tissue in the seated position and to measure differences in soft tissue between loaded and unloaded conditions. Methods: The right gluteal region of able-bodied participants (18--50 years old) was scanned with US (8--10 MHz linear probe, GE LOQIQ 6) in loaded- and unloaded-seated positions on two days. In the loaded-seated position, the soft tissue below the peak of the ischial tuberosity was scanned with subjects seated upright on a custom chair that included a gel pad embedded in the seat cushion and Plexiglas. The tissue in the unloaded-seated position was scanned with subjects lying in a lateral decubitus position in hip (105°) and knee flexion (90°). Total tissue, skin+adipose and muscle+tendon was measured. Intraclass correlations (ICC) were used to measure test-retest reliability. Paired t-test was used to compare soft tissue compression of muscle+tendon vs skin+adipose with significance at p<0.05. Results: Eleven males (BMI: 24±4 kg/m²) and 13 females (BMI: 23±4 kg/m²) were studied. Reliability was excellent between days (ICC=0.941--0.981) in all regions/conditions except for seatedloaded muscle+tendon (ICC= 0.884). However, males had excellent reliability for muscle+tendon (ICC= 0.919). The muscle+ tendon was compressed more (71.0±13.2%) compared to skin+ adipose (40.9±21.8%, p<0.001). Conclusion: Overall, soft tissue of the buttocks in a seated position was highly reliable with muscle+tendon tissue experiencing greater reductions during loading than skin+adipose. MSU IRB approved study. [ABSTRACT FROM AUTHOR]