Acupuncture needle manipulation has been previously shown to result in measurable changes in connective tissue architecture in animal experiments. In this study, we used a novel in vivo ultrasound (US)-based technique to quantify tissue displacement during acupuncture manipulation in humans. B-scan ultrasonic imaging was performed on the thighs of 12 human subjects at different stages of needle motion, including varying amounts of rotation, downward and upward movement performed with a computer-controlled acupuncture needling instrument. Tissue displacements, estimated using cross-correlation techniques, provided successful mapping and quantitative analysis of spatial and temporal tissue behavior during acupuncture needle manipulation. Increasing amounts of rotation had a significant linear effect on tissue displacement during downward and upward needle motion, as well as on rebound tissue displacement after downward needle movement. In addition to being a valuable tool for studies of acupuncture's mechanism of action, this technique may have applications to other types of needling including biopsies.