Background -Excessive sedentary time is ubiquitous in developed nations and is associated with deleterious health outcomes. Few studies have examined whether the manner in which sedentary time is accrued (in short or long bouts) carries any clinical relevance. The purpose of this study was to examine the association of prolonged-uninterrupted sedentary behavior with glycemic biomarkers in a cohort of U.S. Hispanic/Latino adults. Methods -We studied 12,083 participants from the Hispanic Community Health Study/Study of Latinos (HCHS/SOL), a population-based study of Hispanic/Latino adults 18-74 years. Homeostatic model assessment of insulin resistance (HOMA-IR) and glycosylated hemoglobin (Hb1Ac) were measured from a fasting blood sample and 2-hour glucose was measured following an oral glucose tolerance test. Sedentary time was objectively measured using a hip-mounted accelerometer. Prolonged, uninterrupted sedentariness was expressed as mean sedentary bout length. Results -Adjusted for potential confounders and moderate-vigorous physical activity, longer sedentary bout duration was dose-dependently associated with increased HOMA-IR (p-trend <0.001) and 2-hour glucose levels (p-trend=0.015). These associations were not independent of total sedentary time, however a significant interaction between sedentary bout duration and total sedentary time was observed. Evaluation of the joint association of total sedentary time and sedentary bout duration showed that participants in the upper quartile for both sedentary characteristics (i.e. high total sedentary time and high sedentary bout duration) had the highest levels of HOMA-IR (p<0.001 vs. low group for both sedentary characteristics) and 2-hour glucose (p=0.002 vs. low group for both sedentary characteristics). High total sedentary time or high sedentary bout duration alone were not associated with differences in any glycemic biomarkers. Conclusions -Accruing sedentary time in prolonged, uninterrupted bouts may be deleteriously associated with biomarkers of glucose regulation.