Aging-related decline in functional mobility is associated with loss of independence. This decline may be mitigated through programs of physical activity. Despite reports of aging-related mobility impairment in middle-aged adults, this age group has been largely overlooked in terms of exercise programs that target functional mobility and the preservation of independence in older age. A method to quantitatively assess changes in functional mobility could direct rehabilitation in a proactive rather than reactive manner. Thirty-three healthy but sedentary middle-aged adults participated in a four week low-volume, vigorous intensity stepping exercise program. Two baseline testing sessions and one post-training testing session were conducted. Functional mobility was assessed using the timed up and go (TUG) test, with its constituent sit-to-walk and walk-to-sit phases examined using a novel inertial sensor-based method. Additionally, semi-tandem balance and knee extensor muscle isometric torque were assessed. Trunk acceleration during walk-to-sit reduced significantly post-training, suggesting altered movement control due to the exercise program. No significant training-induced changes in sit-to-walk acceleration, TUG time, balance or torque were observed. The novel method of functional mobility assessment presented provides a reliable means to quantify subtle changes in mobility during postural transitions. Over time, this exercise program may improve functional mobility.