Background: To examine sedentary time, prolonged sedentary bouts and physical activity in Australian employees from different workplace settings, within work and non-work contexts. Methods: A convenience sample of 193 employees working in offices (131), call centres (36) and customer service (26) was recruited. Actigraph GT1M accelerometers were used to derive percentages of time spent sedentary (<100 counts per minute; cpm), in prolonged sedentary bouts (≥20 minutes or ≥30 minutes), light-intensity activity (100–1951 cpm) and moderate-to-vigorous physical activity (MVPA; ≥1952 cpm). Using mixed models adjusted for confounders, these were compared for: work days versus non-work days; work hours versus non-work hours (work days only); and, across workplace settings. Results: Working hours were mostly spent sedentary (77.0%, 95%CI: 76.3, 77.6), with approximately half of this time accumulated in prolonged bouts of 20 minutes or more. There were significant (p<0.05) differences in all outcomes between workdays and non-work days, and, on workdays, between work- versus non-work hours. Results consistently showed “work” was more sedentary and had less light-intensity activity, than “non-work”. The period immediately after work appeared important for MVPA. There were significant (p<0.05) differences in all sedentary and activity outcomes occurring during work hours across the workplace settings. Call-centre workers were generally the most sedentary and least physically active at work; customer service workers were typically the least sedentary and the most active at work. Conclusion: The workplace is a key setting for prolonged sedentary time, especially for some occupational groups, and the potential health risk burden attached requires investigation. Future workplace regulations and health promotion initiatives for sedentary occupations to reduce prolonged sitting time should be considered.

Sleep and sedentary and active behaviors are linked to cardiovascular disease risk biomarkers, and across a 24-hour day, increasing time in 1 behavior requires decreasing time in another. We explored associations of reallocating time to sleep, sedentary behavior, or active behaviors with biomarkers. Data (n = 2,185 full sample; n = 923 fasting subanalyses) from the cross-sectional 2005–2006 US National Health and Nutrition Examination Survey were analyzed. The amounts of time spent in sedentary behavior, light-intensity activity, and moderate-to-vigorous physical activity (MVPA) were derived from ActiGraph accelerometry (ActiGraph LLC, Pensacola, Florida), and respondents reported their sleep duration. Isotemporal substitution modeling indicated that, independent of potential confounders and time spent in other activities, beneficial associations (P < 0.05) with cardiovascular disease risk biomarkers were associated with the reallocation of 30 minutes/day of sedentary time with equal time of either sleep (2.2% lower insulin and 2.0% lower homeostasis model assessment of β-cell function), light-intensity activity (1.9% lower triglycerides, 2.4% lower insulin, and 2.2% lower homeostasis model assessment of β-cell function), or MVPA (2.4% smaller waist circumference, 4.4% higher high-density lipoprotein cholesterol, 8.5% lower triglycerides, 1.7% lower glucose, 10.7% lower insulin, and 9.7% higher homeostasis model assessment of insulin sensitivity. These findings provide evidence that MVPA may be the most potent health-enhancing, time-dependent behavior, with additional benefit conferred from light-intensity activities and sleep duration when reallocated from sedentary time.