Background According to cross-sectional and acute experimental evidence, reducing sitting time should improve cardiometabolic health risk biomarkers. Furthermore, the improvements obtained may depend on whether sitting is replaced with standing or ambulatory activities. Based on data from the Stand Up Victoria multicomponent workplace intervention, we examined this issue using compositional data analysis - a method that can examine and compare all activity changes simultaneously. Methods Participants receiving the intervention (n = 136 ≥ 0.6 full-time equivalent desk-based workers, 65% women, mean ± SD age = 44.6 ± 9.1 yr from seven worksites) were asked to improve whole-of-day activity by standing up, sitting less, and moving more. Their changes in the composition of daily waking hours (activPAL-assessed sitting, standing, and stepping) were quantified then tested for associations with concurrent changes in cardiometabolic risk (CMR) scores and 14 biomarkers concerning body composition, glucose, insulin, and lipid metabolism. Analyses were by mixed models, accounting for clustering (3 months, n = 105-120; 12 months, n = 80-97). Results Sitting reduction was significantly (P < 0.05) associated only with lower systolic blood pressure at 3 months, and with CMR scores, weight, body fat, waist circumference, diastolic blood pressure, and fasting triglycerides, total/HDL cholesterol, and insulin at 12 months. Significant differences between standing and stepping were only observed for systolic blood pressure and insulin; both favored stepping. However, replacing sitting with standing was significantly associated only with improvements in CMR scores, whereas replacing sitting with stepping was significantly associated with CMR scores and six biomarkers. Conclusions Improvements in several cardiometabolic health risk biomarkers were significantly associated with sitting reductions that occurred in a workplace intervention. The greatest degree and/or widest range of cardiometabolic benefits appeared to occur with long-term changes, and when increasing ambulatory activities. Trial Registration: ACTRN1211000742976.