Background: To determine whether alternating bouts of sitting and standing at work influences daily workplace energy expenditure (EE). Methods: Twenty-three overweight/obese office workers (mean ± SD; age: 48.2 ± 7.9 y, body mass index: 29.6 ± 4.0 kg/m2) undertook two 5-day experimental conditions in an equal, randomized order. Participants wore a "metabolic armband" (SenseWear Armband Mini) to estimate daily workplace EE (KJ/8 h) while working (1) in a seated work posture (SIT condition) or (2) alternating between a standing and seated work posture every 30 minutes using a sit-stand workstation (STAND-SIT condition). To assess the validity of the metabolic armband, a criterion measure of acute EE (KJ/min; indirect calorimetry) was performed on day 4 of each condition. Results: Standing to work acutely increased EE by 0.7 [95% CI 0.3-1.0] KJ/min (13%), relative to sitting (P =.002). Compared with indirect calorimetry, the metabolic armband provided a valid estimate of EE while standing to work (mean bias: 0.1 [-0.3 to 0.4] KJ/min) but modestly overestimated EE while sitting (P =.005). Daily workplace EE was greatest during the STAND-SIT condition (mean condition difference [95% CI]: 76 [8-144] KJ/8-h workday, P =.03). Conclusions: Intermittent standing at work can modestly increase daily workplace EE compared with seated work in overweight/obese office workers.