Background & aims: Physical activity (PA) breaks may effectively attenuate the detrimental impact of prolonged sitting on acute cognitive performance, perceivable benefits (e.g. mood), vascular function, and metabolic health. To date, the impact of meal composition on the effects of sedentary behavior and/ or PA breaks on health has been scarcely studied. Therefore, our aim was to investigate whether meal composition alters how sedentary behavior and PA breaks affect these acute health outcomes. Methods: A total of 24 overweight and obese, sedentary adults completed four conditions in randomized order in a cross-over design: [a] high-protein, low-fat breakfast (HPLF) þ 4hrs uninterrupted sitting (SIT), [b] HPLF þ 4hrs interrupted sitting (ACT; 5-min cycling every 30 min), [c] Western breakfast (WEST; higher in fats/simple sugars, lower in protein/fiber) þ SIT, [d] WEST þ ACT. WEST and HPLF were isocaloric. Linear mixed models were used to examine changes in cognitive performance (Test of Attentional Performance), perceivable benefits (Likert-scales, Profile of Mood States questionnaire), vascular health (carotid artery reactivity, blood pressure), and metabolic health (post-breakfast glucose, insulin, lipids). Results: Independent of meal composition, we did not observe any effect of PA breaks on cognitive performance, vascular health and post-breakfast lipid responses. PA breaks delayed post-breakfast mood and vigor decrements, as well as increases in fatigue and sleepiness (all p < 0.05), but effects were independent of meal composition (p > 0.05). WEST resulted in higher post-breakfast glucose levels compared to HPLF (p < 0.05), while PA breaks did not impact this response (p > 0.05). PA breaks reduced post-breakfast insulin (p < 0.05), which did not differ between meals (p > 0.05). Conclusions: The acute impact of PA breaks and/or prolonged sitting on cognitive performance, perceivable benefits, and vascular and metabolic health was not altered by the composition of a single meal in overweight/obese, sedentary adults. Possibly, breaking up prolonged sitting, rather than meal composition, is a more potent strategy to impact acute health outcomes, such as perceivable benefits and insulin levels.