Acknowledging the detrimental effects of prolonged sitting, this studyexamined the effects of an acute exercise break during prolonged sitting onexecutive function, cortical hemodynamics, and microvascular status. In thisrandomized crossover study, 71 college students completed three conditions:(i) uninterrupted sitting (SIT); (ii) SIT with a 15 min moderate-intensity cyclingbreak (MIC); and (iii) SIT with a 15 min vigorous-intensity cycling break (VIC).Behavioral outcomes, retinal vessel diameters (central retinal arteryequivalents [CRAE], retinal vein equivalents [CRVE], arteriovenous ratio [AVR]),cortical activation, and effective connectivity were evaluated. Linear mixedmodels identified significant positive effects of exercise conditions onbehavioral reaction time (RT), error rate, and inverse efficiency score(𝜷 = −2.62, −0.19, −3.04: ps < 0.05). MIC and VIC conditions producedpre-to-post-intervention increases in CRAE and CRVE (𝜷 = 4.46, 6.34), frontalactivation, and resting-state and task-state causal density (𝜷 = 0.37, 0.06)(ps < 0.05) compared to SIT; VIC was more beneficial for executive functionand neurobiological parameters. The effect of AVR on average RT wasmediated through task-based causal density (indirect effect: −0.82). Acutelyinterrupting prolonged sitting improves executive function, microvascularstatus, and cortical activation and connectivity, with causal density mediatingthe microvascular-executive function link.