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This study aimed to experimentally determine whether cardiorespiratory fitness (CRF) modifies postprandial glycemia during prolonged sitting and investigated the potentially blunting influence this may have on the benefits of interrupting postprandial sitting time with light activity breaks.Thirty-four adults (18 women; 16 men; mean ± SD age, 40 ± 9 yr, body mass index, 24.5 ± 3 kg·m−2) undertook two 7.5-h experimental conditions in a randomized order: 1) Prolonged sitting; 2) Sitting interspersed with 5 min light walking bouts every 30 min. Blood samples were obtained while fasting and postprandially after ingestion of two identical meals. Incremental area under the curve (iAUC) was calculated for glucose and insulin throughout experimental conditions. Maximal exercise testing quantified peak oxygen consumption (V˙O2 peak) as a measure of CRF. A repeated-measures ANOVA investigated whether V˙O2 peak modified glucose and insulin iAUC between conditions.Breaking sedentary time with light walking breaks reduced blood glucose iAUC from 3.89 ± 0.7 to 2.51 ± 0.7 mmol·L−1·h−1 (P = 0.015) and insulin iAUC from 241 ± 46 to 156 ± 24 mU·L−1·h−1 (P = 0.013) after adjustment for V˙O2 peak and sex. A significant interaction between treatment response and V˙O2 peak was observed for glucose (P = 0.035), but not insulin (P = 0.062), whereby the treatment effect reduced with higher CRF. Average blood glucose iAUC responses for a man at the 25th centile of CRF within our cohort (42.5 mL·kg−1·min−1) went from 5.80 to 2.98 mmol·L−1·h−1 during the prolonged sitting and light walking break conditions respectively, whereas average responses for a man at the 75th centile of CRF (60.5 mL·kg−1·min−1) went from 1.99 to 1.78 mmol·L−1·h−1. Similar trends were observed for women.Individuals with low CRF gained the most metabolic benefit from breaking prolonged sitting with regular bouts of light walking.