Breaking up prolonged sitting has been beneficially associated with cardio-metabolic risk markers in both observational and intervention studies. We aimed to define the acute transcriptional events induced in skeletal muscle by breaks in sedentary time. Overweight/obese adults participated in a randomized three-period, three-treatment cross-over trial in an acute setting. The three 5 hour interventions were performed in the post-prandial state after a standardized test drink and included: seated position with no activity, seated with 2-minute bouts of light- or moderate-intensity treadmill walking every 20 minutes. Vastus lateralis biopsies were obtained in 8 participants after each treatment and gene expression examined using microarrays validated with real-time qPCR. There were 75 differentially-expressed genes between the three conditions. Pathway analysis indicated the main biological functions affected were related to small molecule biochemistry, cellular development, growth and proliferation and carbohydrate metabolism. Interestingly, differentially expressed genes were also linked to cardiovascular disease. For example, relative to prolonged sitting, activity bouts increased expression of Nicotamide N-methyltransferase (NNMT), which modulates anti-inflammatory and anti-oxidative pathways and triglyceride metabolism. Activity bouts also altered expression of 10 genes involved in carbohydrate metabolism including increased expression of dynein light chain (DYNLL1), which may regulate translocation of the GLUT4 glucose transporter. In addition, breaking up sedentary time reversed the effects of chronic inactivity on expression of some specific genes. This study provides insight into the muscle regulatory systems and molecular processes underlying the physiological benefits induced by interrupting prolonged sitting.