Background: A single bout of moderate-to-vigorous intensity exercise improves both metabolic and cognitive function. In addition to exercise, emerging evidence suggests that reducing sitting time may be another strategy for improving metabolic function. However, the combined effects of acute exercise with reductions in sitting time on cognitive and metabolic function are largely unknown. Methods/design: This is a dual-site randomised crossover trial involving three acute experimental conditions separated by a minimum six-day washout period. This trial included physically inactive and sedentary older adults (55–80 years) who were overweight to obese (body mass index 25–45 kg/m2). Participants were recruited to complete the following eight hour laboratory-based conditions in a random order; 1) Sitting: uninterrupted sitting (8hrs, control); 2) Exercise: sitting (1hr), moderate intensity walking (30mins) followed by uninterrupted sitting (6.5hrs); 3) Exercise + Breaks: sitting (1hr), moderate intensity walking (30mins) followed by sitting (6.5hrs) interrupted every 30 minutes with three minutes of light intensity walking. The primary outcome will be cognitive function, assessed using a battery of memory tests (Cogstate). Secondary outcome measures will include postprandial glucose, insulin, triglycerides, brain derived neurotrophic factor (BDNF), catecholamines, brain blood flow, brachial artery flow mediated dilation (FMD) and blood pressure. Discussion: This evidence will inform practical preventive strategies aimed at optimising daily cognitive, vascular and metabolic function among the large number of older adults who are behaviourally exposed to prolonged uninterrupted sitting.