Background: Sedentary activity and sitting for at least 10 hours per day can increase the risk for cardiovascular disease by more than 60%. Use of standing desks may decrease sedentary time and improve cardiovascular health. Acute standing lowers pulse wave velocity (PWV), but chronic effects remain unknown. The purpose of this study was to determine the effect of chronic standing desk use on arterial stiffness versus seated controls. Methods: A total of 48 adults participated in this study. Twenty-four participants qualified as seated desk users (age 41  y, body mass index 25  kg/m2) and 24 as standing desk users (age 45  y, body mass index 25  kg/m2). Arterial stiffness was assessed as PWV within the aorta, arm, and leg. Results: Carotid–femoral PWV (cfPWV) was not different between seated (6.6 [1.3] m/s) and standing (6.9 [1.3] m/s) groups (P = .47). Similarly, there were no differences in arm or leg PWV between groups (P = .13 and P = .66, respectively). A secondary analysis of traditional factors of age and aerobic fitness revealed significant differences in cfPWV in seated and standing desk participants. Age also significantly influenced cfPWV across conditions. Conclusions: Standing for >50% of a workday did not affect PWV. Consistent with previous research, fitness and age are important modulators of arterial stiffness.