The effect of instrument-assisted soft tissue mobilization (ISTM) on passive properties and inflammation in human skeletal muscle has not been evaluated. Passive properties of muscle, inflammatory myokines and subjective reporting of functional ability were used to identify the effects of ISTM on the plantar flexors. 11 healthy men were measured for passive musculotendinous stiffness (MTS), passive range of motion (PROM), passive resistive torque (PASTQ) and maximum voluntary contraction peak torque (MVCPT) for plantar flexor muscles of the lower leg. Interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) were measured from muscle biopsies from the gastrocnemius, and subjective measurements of functional ability were taken using the perception of functional ability questionnaire (PFAQ). MTS, PROM, PRT and MVCPT were measured in the treatment leg (TL) and control leg (CL) before, immediately after, 24 h, 48 h and 72 h following IASTM. Biopsies for IL-6 and TNF-α and PFAQ responses were collected before as well as 24 h, 48 h and 72 h after IASTM. There were no significant differences in MTS, PROM, PASTQ, MVCPT, IL-6 and TNF-α between the TL or CL. A significant decrease in the perception of function and a significant increase in pain for the TL were found following IASTM