Excessive time spent in sedentary behaviours (sitting or lying with low energy expenditure) is associated with an increased risk for type 2 diabetes, cardiovascular disease and some cancers. Desk-based office workers typically accumulate high amounts of daily sitting time, often in prolonged unbroken bouts. The Stand Up Victoria study aims to determine whether a 3-month multi-component intervention in the office setting reduces workplace sitting, particularly prolonged, unbroken sitting time, and results in improvements in cardio-metabolic biomarkers and work-related outcomes, compared to usual practice. A two-arm cluster-randomized controlled trial (RCT), with worksites as the unit of randomization, will be conducted in 16 worksites located in Victoria, Australia. Work units from one organisation (Department of Human Services, Australian Government) will be allocated to either the multi-component intervention (organisational, environmental [height-adjustable workstations], and individual behavioural strategies) or to a usual practice control group. The recruitment target is 160 participants (office-based workers aged 18–65 years and working at least 0.6 full time equivalent) per arm. At each assessment (0- [baseline], 3- [post intervention], and 12-months [follow-up]), objective measurement via the activPAL3 activity monitor will be used to assess workplace: sitting time (primary outcome); prolonged sitting time (sitting time accrued in bouts of ≥30 minutes); standing time; sit-to-stand transitions; and, moving time. Additional outcomes assessed will include: non-workplace activity; cardio-metabolic biomarkers and health indicators (including fasting glucose, lipids and insulin; anthropometric measures; blood pressure; and, musculoskeletal symptoms); and, work-related outcomes (presenteeism, absenteeism, productivity, work performance). Incremental cost-effectiveness and identification of both workplace and individual-level mediators and moderators of change will also be evaluated. Stand Up Victoria will be the first cluster-RCT to evaluate the effectiveness of a multi-component intervention aimed at reducing prolonged workplace sitting in office workers. Strengths include the objective measurement of activity and assessment of the intervention on markers of cardio-metabolic health. Health- and work-related benefits, as well as the cost-effectiveness of the intervention, will help to inform future occupational practice. ACTRN1211000742976