Purpose The purpose of this preliminary study was to compare the effects of 1-h sitting with and without short active breaks on muscle stiffness as measured by shear-wave elastography (SWE). Methods The participants (7 females, 3 males; age: 24.9 ± 1.2 years) completed two (with and without active breaks) 1-h sitting exposures on separate days. Active breaks (2–3 min) were performed at 20 min and 40 min time marks and comprised simple stretching and activation exercises. Before, during (30 min) and after (1 h) of sitting, shear modulus of upper trapezius, lumbar region of erector spinae and rectus femoris muscles was measured with SWE. Results Statistically significant effects of sitting exposure in erector spinae muscle stiffness were noted (p = 0.041; η2 = 0.38). There were no other statistically significant effects of sitting exposure or condition (with/without breaks). Conclusions Although few statistically significant effects were detected, the trends in this preliminary trial suggest that prolonged sitting increases muscle stiffness and warrants further investigation of short active breaks with larger sample sizes.