Objective: To quantify the association between daily total sitting and all-cause mortality risk and to examine dose-response relationships with and without adjustment for moderate-to-vigorous physical activity. Methods: Studies published from 1989 to January 2013 were identified via searches of multiple databases, reference lists of systematic reviews on sitting and health, and from authors' personal literature databases. We included prospective cohort studies that had total daily sitting time as a quantitative exposure variable, all-cause mortality as the outcome and reported estimates of relative risk, or odds ratios or hazard ratios with 95% confidence intervals. Two authors independently extracted the data and summary estimates of associations were computed using random effects models. Results: Six studies were included, involving data from 595,086 adults and 29,162 deaths over 3,565,569 person-years of follow-up. Study participants were mainly female, middle-aged or older adults from high-income countries; mean study quality score was 12/15 points. Associations between daily total sitting time and all-cause mortality were not linear. With physical activity adjustment, the spline model of best fit had dose-response HRs of 1.00 (95% CI: 0.98-1.03), 1.02 (95% CI: 0.99-1.05) and 1.05 (95% CI: 1.02-1.08) for every 1-hour increase in sitting time in intervals between 0-3, >3-7 and >7 h/day total sitting, respectively. This model estimated a 34% higher mortality risk for adults sitting 10 h/day, after taking physical activity into account. The overall weighted population attributable fraction for allcause mortality for total daily sitting time was 5.9%, after adjusting for physical activity. Conclusions: Higher amounts of daily total sitting time are associated with greater risk of all-cause mortality and moderate-to-vigorous physical activity appears to attenuate the hazardous association. These findings provide a starting point for identifying a threshold on which to base clinical and public health recommendations for overall sitting time, in addition to physical activity guidelines. © 2013 Chau et al.