The negative effect of sedentary behaviour on type 2 diabetes markers is established, but the interaction with measures of physical activity is still largely unknown. Previous studies have analysed associations with single-activity models, which ignore the interaction with other behaviours. By including results from various analytical approaches, this review critically summarises the effects of sedentary behaviour on diabetes markers and the benefits of substitutions and compositions of physical activity. Ovid Medline, Embase and Cochrane Library databases were systematically searched. Studies were selected if sedentary behaviour and physical activity were measured by accelerometer in the general population, and if associations were reported with glucose, insulin, HOMA-IR, insulin sensitivity, HbA1c, diabetes incidence, CRP and IL-6. Forty-five studies were included in the review. Conclusive detrimental associations with sedentary behaviour were determined for 2-h insulin (6/12 studies found associations), fasting insulin (15/19 studies), insulin sensitivity (4/6 studies), diabetes (3/4 studies) and IL-6 (2/3 studies). Reallocating sedentary behaviour to light or moderate-to-vigorous activity has a beneficial effect for 2-h glucose (1/1 studies), fasting insulin (3/3 studies), HOMA-IR (1/1 studies) and insulin sensitivity (1/1 studies). Compositional measures of sedentary behaviour were found to affect 2-h glucose (1/1 studies), fasting insulin (2/3 studies), 2-h insulin (1/1 studies), HOMA-IR (2/2 studies) and CRP (1/1 studies). Different analytical methods produced conflicting results for fasting glucose, 2-h glucose, 2-h insulin, insulin sensitivity, HOMA-IR, diabetes, hbA1c, CRP and IL-6. Studies analysing data by quartiles report independent associations between sedentary behaviour and fasting insulin, HOMA-IR and diabetes only for high duration of sedentary time (7-9 hours/day). However, this review could not provide sufficient evidence for a time-specific cut-off of sedentary behaviour for diabetes biomarkers. While substituting sedentary behaviour with moderate-to-vigorous activity brings greater improvements for health, light activity also benefits metabolic health. Future research should elucidate the effects of substituting and combining different activity durations and modalities.