Background Emerging evidence suggests accruing sedentary behavior (SB) in relatively more prolonged periods may convey additional cardiometabolic risks, but few studies have examined prospective outcomes. We examined the association of SB accumulation patterns with incident cardiovascular disease (CVD), cancer, and all-cause mortality (ACM). Methods and Results Data were from 7671 EPIC-Norfolk (European Prospective Investigation Into Cancer and Nutrition-Norfolk) cohort middle- to older-aged adults who wore accelerometers on the right hip for 4 to 7 days. Cox proportional hazards regression modeled associations between 2 measures of SB accumulation and incident CVD, cancer, and ACM. These were usual SB bout duration (the midpoint of each individual's SB accumulation curve, fitted using nonlinear regression) and alpha (hybrid measure of bout frequency and duration, with higher values indicating relatively shorter bouts and fewer long bouts). Models were adjusted for potential confounders, then further for 24-hour time-use compositions. During mean follow-up time of 6.4 years, 339 ACM, 1106 CVD, and 516 cancer events occurred. Elevated rates of incident cancer and ACM were seen with more prolonged SB accumulation (lower alpha, higher usual SB bout duration) but not CVD. For usual SB bout duration and alpha, respectively, the confounder-adjusted hazard ratios per SD of the exposure were 1.12 (95% CI, 1.02-1.23) and 0.88 (95% CI, 0.79-0.98) with incident cancer and 1.16 (95% CI, 1.07-1.26) and 0.80 (95% CI, 0.72-0.89) with ACM (all P<0.05). Further adjustment for 24-hour time use weakened associations with ACM for usual bout duration (hazard ratio, 1.06; 95% CI, 0.97-1.16; P=0.209) and partially for alpha (hazard ratio, 0.87; 95% CI, 0.77-0.99; P=0.029). Conclusions Accruing SB in longer bout durations was associated with higher rates of incident cancer and ACM but not with incident CVD, with some evidence of direct SB accumulation effects independent of 24-hour time use. Findings provide some support for considering SB accumulation as an adjunct target of messaging to "sit less and move more."