This study was a randomised, three-period, three-treatment crossover trial to examine the acute effects of regularly breaking up seated office work with short bouts of standing or light-intensity walking on postprandial interstitial glucose concentration. Seventeen middle-aged office workers performed three five-hour trial conditions at their workplace in a random order: 1) uninterrupted sitting; 2) sitting interrupted by two minutes of standing every 20 minutes; and 3) sitting interrupted by two minutes of light-intensity walking every 20 minutes. Participants consumed two standardised test drinks at the start of each trial condition and average interstitial glucose concentration was recorded every five minutes for the duration of the study using a continuous glucose monitor. The study found that five-hour interstitial glucose incremental area under the curve (iAUC) was 55.5% lower after sitting interrupted by light-intensity walking compared with after uninterrupted sitting, indicating that regularly breaking up prolonged sitting lowers postprandial glycemia in middle-aged adults without metabolic impairment.