Background Sedentary behaviour is associated with impaired cognition, whereas exercise can acutely improve cognition. Objective We compared the effects of a morning bout of moderate-intensity exercise, with and without subsequent light-intensity walking breaks from sitting, on cognition in older adults. Methods Sedentary overweight/obese older adults with normal cognitive function (n=67, 67±7 years, 31.2±4.1 kg/m 2) completed three conditions (6-day washout): SIT (sitting): uninterrupted sitting (8 hours, control); EX+SIT (exercise + sitting): sitting (1 hour), moderate-intensity walking (30 min), uninterrupted sitting (6.5 hours); and EX+BR (exercise + breaks): sitting (1 hour), moderate-intensity walking (30 min), sitting interrupted every 30 min with 3 min of light-intensity walking (6.5 hours). Cognitive testing (Cogstate) was completed at four time points assessing psychomotor function, attention, executive function, visual learning and working memory. Serum brain-derived neurotrophic growth factor (BDNF) was assessed at six time points. The 8-hour net area under the curve (AUC) was calculated for each outcome. Results Working memory net AUC z-score·hour (95% CI) was improved in EX+BR with a z-score of +28 (-26 to +81), relative to SIT, -25 (-79 to +29, p=0.04 vs EX+BR). Executive function net AUC was improved in EX+SIT, -8 (- 71 to +55), relative to SIT, -80 (-142 to -17, p=0.03 vs EX+SIT). Serum BDNF net AUC ng/mL·hour (95% CI) was increased in both EX+SIT, +171 (-449 to +791, p=0.03 vs SIT), and EX+BR, +139 (-481 to +759, p=0.045 vs SIT), relative to SIT, -227 (-851 to +396). Conclusion A morning bout of moderate-intensity exercise improves serum BDNF and working memory or executive function in older adults, depending on whether or not subsequent sitting is also interrupted with intermittent light-intensity walking. Trial registration number ACTRN12614000737639.