Sedentary behavior (SB) negatively affects office workers health, besides adverse effects on well-being and productivity. Portugal has the lowest levels of physical activity (PA) in Europe and high levels of SB. This study aimed to analyze the effect of a 6-month multi-component intervention to decrease SB and improve PA, on perceived musculoskeletal symptoms in the office work. This study included an intervention group (n = 40) and a control group (n = 40). To measure the SB/PA levels, ActiGraph WGT3X-BT was used. To evaluate musculoskeletal symptoms, Nordic Musculoskeletal Questionnaire was applied. The intervention decreased SB (p = 0.017) and improved moderate to vigorous physical activity (p = 0.021) in the intervention group. In conclusion, the results showed that intervention could increase physical activity and decrease sedentary behavior in office workers.