Recent updates to national physical activity guidelines now highlight the importance of reducing sedentary time. However, at present only general recommendations are possible (i.e. “Sit less, move more”). There remains a need to investigate the strength, temporality, specificity and dose-response nature of sedentary behaviour associations with chronic disease, along with potential underlying mechanisms. Stemming from a recent research workshop organised by the Sedentary Behaviour Council themed ‘‘Sedentary behaviour mechanisms—biological and behavioural pathways linking sitting to adverse health outcomes”, this paper aims to: 1) discuss existing challenges and scientific discussions within this advancing area of science, 2) highlight and discuss emerging areas of interest, and 3) point to pertinent future directions. A brief knowledge update is provided, reflecting upon current and evolving thinking/discussions, and the rapid accumulation of new evidence linking sedentary behaviour to chronic disease. Succinct research 'action-points' are made at the end of each section – spanning from measurement systems and analytic methods, genetic epidemiology, causal mediation and experimental studies, to biological and behavioural determinants and mechanisms. A better understanding of whether and how sedentary behaviour is causally related with chronic disease will allow for more meaningful conclusions in the future and assist in refining both clinical and public health policies/recommendations.