Purpose To investigate how the pattern of sedentary behaviour affects intra-day glucose regulation in type 2 diabetes. Methods This intensive longitudinal study was conducted in 37 participants with type 2 diabetes (age, 62.8 ± 10.5 years). Glucose and sedentary behaviour/physical activity were assessed with a continuous glucose monitoring (Abbott FreeStyle Libre) and an activity monitor (activPAL3) for 14 days. Multiple regression models with generalised estimating equations (GEEs) approach were used to assess the associations of sedentary time and breaks in sedentary time with pre-breakfast glucose, pre-lunch glucose, pre-dinner glucose, post-breakfast glucose, post-lunch glucose, post-dinner glucose, bedtime glucose, the dawn phenomenon, time in target glucose range (TIR, glucose 3.9-10 mmol/L) and time above target glucose range (TAR, glucose > 10 mmol/L). Results Sedentary time was associated with higher pre-breakfast glucose (p = 0.001), pre-dinner glucose (p < 0.001), postlunch glucose (p = 0.005), post-dinner glucose (p = 0.013) and the dawn phenomenon (p < 0.001). Breaks in sedentary time were associated with lower pre-breakfast glucose (p = 0.023), pre-dinner glucose (p = 0.023), post-breakfast glucose (p < 0.001) and the dawn phenomenon (p = 0.004). The association between sedentary time and less TIR (p = 0.022) and the association between breaks in sedentary time and more TIR (p = 0.001) were also observed. Conclusions Reducing sedentary time and promoting breaks in sedentary time could be clinically relevant to improve intraday glucose regulation in type 2 diabetes. Keywords Type 2 diabetes • Glucose targets • Glycaemic control • Sedentary behaviour • Physical activity • Breaks in sedentary time Abbreviations BMI Body mass index CGM Continuous glucose monitoring GEEs Generalised estimating equations HbA1c Glycated haemoglobin IDF International Diabetes Federation MVPA Moderate to vigorous physical activity QIC Quasi-likelihood under the independence model criterion TIR Time in target glucose range TAR Time above target glucose range * Aye C.