BACKGROUND The aims of this study were to (1) calculate the correlation between different tensile force levels and corresponding muscle stiffness both in vitro and in vivo; (2) determine whether muscle stiffness assessed using a MyotonPRO myotonometer can be used to accurately estimate muscle activity level; and (3) evaluate the inter-operator reliability of MyotonPRO-based measurement in assessing biceps brachii muscle (BBM) stiffness. MATERIAL AND METHODS In Experiment I, muscle stiffness, as measured using the MyotonPRO, was obtained at 0 N, 2 N, 4 N, 6 N, 8 N, and 10 N of applied force on 6 fresh medial gastrocnemius muscle specimens. In Experiment II, 11 healthy subjects were recruited. BBM stiffness, assessed by the same device, was obtained at different tensile force levels, from 0 to 50% of maximal voluntary contraction (MVC). For the reliability test, the score for each subject was quantified by 2 operators (I and II), thrice, at 30-minute intervals on the same day. RESULTS A strong correlation was found between the different tensile force levels, which corresponded to muscle stiffness in vitro (r=0.71-0.95, all P<0.05). In vivo, muscle stiffness increased linearly with an increase of the tensile force levels from 0 to 50% of MVC (r=0.99, P=0.00) and there was a significant difference in BBM stiffness among the incremental isometric tasks (F [1.76, 17.60]=91.52, P=0.00). The inter-operator reliability for the measurement of BBM stiffness was good (ICC=0.86). CONCLUSIONS Our findings indicate that muscle stiffness measured using the MyotonPRO is strongly related to muscle activity level and that the MyotonPRO is a feasible tool for quantifying BBM stiffness as well as for quantifying changes in MVC levels.