Low levels of physical activity (PA) not only increase healt h risks but also affect employee productivity. Although daily activity is interdependent with work productivity and personal health, few studies have examined how substituting physical activities would affect health and work efficiency. The present study aimed to investigate how substituting sedentary behaviors (SB) with increased PA and increasing the intensity of low-level activities during waking times affects the body fat mass and work efficiency of employees. Data were collected from 224 Japanese employees. SB, light physical activity (LPA), moderate physical activity (MPA), and vigorous physical activity (VPA) were measured using a tri-axial accelerometer, and body fat mass and work efficiency were also assessed. Analyses of the effects of substituting behaviors were based on the methods used in the isotemporal substitution model. Body fat mass decreased after substituting behaviors for 30 min per day: from SB to VPA (β = −4.800, 95% CI = −7.500; −2.100), from LPA to VPA (β = −4.680, 95% CI = −7.350; −1.980), and from MPA to VPA (β = −4.920, 95% CI = −7.680; −2.190). For work efficiency and physical activities, a higher work efficiency score was observed when substituting SB with LPA (β = 0.120, 95% CI = 0.030; 0.240), and a lower work efficiency score was observed when substituting LPA with VPA (β = −0.660, 95% CI = −1.350; −0.030). These results should help achieve greater results in promoting health and increasing work productivity by properly distributing and practicing daily physical activities during work hours.