Background: Levels of sitting among adolescents are high, especially during the school day. The acute cognitive and health consequences associated with prolonged sitting are poorly understood in adolescents. This randomized crossover design study examined the acute effects of a simulated school day with reduced sitting or usual sitting on adolescents' cognitive function and cardiometabolic biomarkers. Methods: Eighteen healthy school aged adolescents were recruited from the community to the study (11 males; 7 females; mean age [SD] = 13.5 ± 0.9 years). Two protocols were developed to simulate an adolescent school day, the amount of time spent sitting was manipulated reflecting: a 'typical' day (65% of the time spent sitting with two sitting bouts sitting > 20 min) and a 'reduced sitting' day (adolescents sat for 50% less time with no bouts of sitting > 20 mins). The order that participants were exposed to each condition was randomized (via random number generator). Participants were not fully blinded as they could observe the difference between conditions. Energy intake and moderate to vigorous physical activity (MVPA) were standardized for both conditions and monitored for 48 h post-condition for compensatory effects. Cognitive (working memory) and cardiometabolic outcomes (lipids, glucose, insulin, IL-6, apo-A1, apo-B, blood pressure,) were assessed pre and post for both conditions, BMI and body fat were assessed on the morning of the intervention. Data were analyzed using linear mixed models. Standardised effect sizes were calculated. Results: Compared with the typical school day, the reduced sitting day demonstrated significant positive effects for apoB/apoA-1 ratio (adjusted difference ± SD) -0.02 ± 0.03; P = 0.03; effect size [Cohen's d] = -0.67. Findings for total cholesterol -0.19 ± 0.27; P = 0.28; d = -0.71; HDL cholesterol -0.23 ± 0.50; P = 0.12 d = -0.66; and total cholesterol/HDL ratio 0.25 ± 0.53; P = 0.25; d = 0.51 and for cognition 0.64 ± 0.15; P = 0.15; d = 0.54 were non-significant. There were no compensatory changes in participant energy expenditure or energy intake for 48 h post intervention. Conclusion: Reducing school day sitting time in adolescents' resulted in significant improvements in apoB/apoA-1 ratio with medium effect sizes for total cholesterol, HDL cholesterol and total cholesterol/HDL ratio. Cognitive function results showed the equivalent of a 6 month improvement in effective mental-attentional capacity. Trial registration: The trial was registered as a clinical trial with the Australian and New Zealand Clinical Trials Registry (ACTRN12614001064695) on the 3rd of October 2014 - registered retrospectively.