OBJECTIVE: To examine the associations of sedentary time and physical activity with biomarkers of cardiometabolic health, including the potential collective impact of shifting mean time use from less- to more-active behaviours (cross-sectionally, using isotemporal substitution), in adults with type 2 diabetes. METHODS: Participants with overweight/obese body mass index (BMI; ≥25 kg/m2) (n = 279; 158 men, mean [SD] age = 58.2 [8.6] years) wore Actigraph GT1M accelerometers (waking hours; seven days) to assess moderate- to vigorous-intensity physical activity (MVPA), light-intensity activity, and sedentary time (segregated into non-prolonged [accumulated in bouts <30min] and prolonged [accumulated in bouts ≥30 min]). Cross-sectional associations with waist circumference, BMI, fasting blood (HbA1c, glucose, triacylglycerols, high-density lipoprotein cholesterol), and blood pressure of these activity variables (30 min/day increments) were examined adjusted for confounders and wear then, if significant, examined using isotemporal substitution modelling. RESULTS: Waist circumference and BMI were significantly (p<0.05) associated with more prolonged sedentary time and less light-intensity activity. Light intensity activity was also significantly associated with lower fasting plasma glucose (relative rate: 0.98, 95% CI: 0.97, 1.00; p<0.05). No biomarker was significantly associated with non-prolonged sedentary time or MVPA. Lower mean prolonged sedentary time (-30 min/day) with higher mean light intensity time (+30 min/day) was significantly associated with lower waist circumference (β = -0.77, 95% CI: -1.33, -0.22 cm). Lower mean prolonged sedentary time (-30 min/day) with either 30 min/day higher mean non-prolonged sedentary time (β = -0.35, 95%CI: -0.70, -0.01 kg/m2) or light-intensity time (β = -0.36, -0.61, -0.11 kg/m2) was associated with significantly lower average BMI. CONCLUSIONS: Significantly improved mean levels of waist circumference and BMI were observed when shifting time from prolonged sedentary to non-prolonged sedentary or light-intensity activity (cross-sectionally). Lifestyle interventions in overweight/obese adults with type 2 diabetes might consider targeting shifts in these non-MVPA activities to more rigorously evaluate their potential cardiometabolic benefit in this population.