Purpose: While merely standing up interrupts sedentary behavior, it is important to study acute metabolic responses during single bouts of sitting and standing to understand the physiological processes affecting the health of office workers. Methods: 18 healthy middle aged women aged 49.4 ± 7.9 years (range: 40 to 64) with a BMI of 23.4 ± 2.8 kg·m -2 volunteered for this laboratory-based randomized crossover trial where they performed two hours desk work either in sitting or standing postures after overnight fasting. Muscle activity (normalized to walking at 5 km/h), respiratory gas exchange and blood samples were assessed following glucose loading (75 g). Results: Compared with seated work, continuous standing resulted in greater activity in the thigh muscles (mean of biceps femoris and vastus lateralis: 17 ± 8% vs. 7 ± 2%, p < 0.001), and leg muscles (mean of tibialis anterior, gastrocnemius medialis and soleus: 16 ± 6% vs. 7 ± 3%, p<0.001), but no increases in back muscle activity (thoracic erector spinae, lumbar erector spinae and multifidus). Concomitant with ~9% higher energy expenditure (EE) (p = 0.002), standing resulted in higher fat oxidation (48 ± 9%EE vs. 39 ± 7%EE, p = 0.008) and lower carbohydrate oxidation (52 ± 9%EE vs. 61 ± 7%EE, p = 0.008) than sitting. Glucose total and net incremental area under the curve were ~10% (p = 0.026) and ~42% (p = 0.017) higher during standing than sitting, respectively. Insulin concentration did not differ between conditions. Conclusion:Compared to sitting, two hours of standing increased muscle activity, fat oxidation and circulating glucose level. These results suggest fuel switching in favor of fat oxidation during standing despite extra carbohydrate availability.