Recent guidelines recommend accruing 2-4h of standing or light activity during the working day. Use of sit-stand desks could achieve this goal, but whether standing can meaningfully increase energy expenditure (EE) is unclear. To study EE, heart rate, feelings and productivity during deskwork while sitting, standing or alternating positions. We measured EE by indirect calorimetry in working adults over three randomly ordered 60-min conditions while performing deskwork: continuous sitting (SIT), 30min of each standing and sitting (STAND-SIT) and continuous standing (STAND). We also assessed heart rate, productivity and self-reported energy, fatigue and pain. Linear mixed models compared minute-by-minute EE and heart rate across conditions. Non-parametric tests compared remaining outcomes across conditions. The study group comprised 18 working adults. Compared with SIT, STAND-SIT engendered an additional 5.5±12.4 kcal/h (7.8% increase) and STAND engendered an additional 8.2±15.9 kcal/h (11.5% increase) (both P < 0.001). Alternating positions to achieve the recommended 4h/day of standing could result in an additional 56.9 kcal/day for an 88.9kg man and 48.3 kcal/day for a 75.5kg woman. STAND-SIT and STAND also increased heart rate over SIT by 7.5±6.8 and 13.7±8.8 bpm, respectively (both P < 0.001). We observed no meaningful differences in feelings or productivity. Desk-based workers could increase EE without added discomfort by using a sit-stand desk. These findings inform future research on sit-stand desks as a part of workplace interventions to increase EE and potentially improve health.