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STUDY DESIGN Randomized clinical trial. BACKGROUND Muscle stiffness is a potential complication after injury and has been shown to be a risk factor for injury in healthy individuals. OBJECTIVES The primary purpose of this study was to assess the short-term effects of manual therapy (MT) on muscle stiffness of the gastrocnemius in both a relaxed and contracted state. The secondary purpose was to assess the reliability of a novel clinical tool (MyotonPRO) to measure muscle stiffness in the gastrocnemius in both a passive and contracted state. METHODS Eighty-four consecutive healthy individuals were randomized to receive Manual Therapy (MT group) directed at the right-side ankle and foot or no treatment (CONTROL group). Muscle stiffness of the gastrocnemius was assessed bilaterally in all participants at baseline and then immediately after intervention in a relaxed and contracted state. Group (MT vs. CONTROL) by side (ipsilateral vs. contralateral) by time (pre vs. post) effects were compared through a 3-way interaction utilizing mixed model ANOVA. Reliability of the MyotonPRO was assessed with two-way mixed model intraclass correlation coefficients. RESULTS There was a significant 3-way interaction for muscle stiffness of the gastrocnemius in a relaxed state (p<0.01), but not contracted state (p=0.54). All conditions had increased resting muscle stiffness from pre to post measures except for the ipsilateral limb of the MT group. There was not a significant interaction for muscle stiffness in a contracted state. Reliability estimates (ICC) for muscle stiffness measures ranged between 0.898 and 0.986. CONCLUSION The change in muscle stiffness of the gastrocnemius in a relaxed state depended upon whether individuals received MT. Muscle stiffness measures were highly reliable based on single measurements. LEVEL OF EVIDENCE Therapy, level 2.