Aims While excessive sitting time is related adversely to cardio-metabolic health, it is unknown whether standing is a suitable replacement activity or whether ambulatory movement is required. Using isotemporal substitution analyses, we modelled cross-sectional associations with cardio-metabolic risk biomarkers of reallocating time (2 h/day) from sitting to standing or to stepping. Methods and results A subsample of participants from the 2011/12 Australian Diabetes, Obesity, and Lifestyle Study wore the posture-based activPAL3 monitor [36-80 years (mean 57.9, SD 9.9 years); 57 women; n = 698 with data]. Associations of activPAL3-derived mean daily time sitting/lying (sitting), standing and stepping with body mass index (BMI), waist circumference, blood pressure, HbA1c, fasting glucose and lipids (high-density lipoprotein-, HDL, and low-density lipoprotein-cholesterol, total/HDL-cholesterol ratio, and triglycerides), and 2-h plasma glucose were examined. Adjusted for relevant confounders, sitting-to-standing reallocations were only significantly (P <0.05) associated with approximately 2 lower fasting plasma glucose, 11 lower triglycerides, 6 lower total/HDL-cholesterol ratio, and 0.06 mmol/L higher HDL-cholesterol per 2 h/day. Sitting-to-stepping reallocations were only significantly associated with approximately 11 lower BMI, 7.5 cm lower waist circumference, 11 lower 2-h plasma glucose, 14 lower triglycerides, and 0.10 mmol/L higher HDL-cholesterol per 2 h/ day, while standing-to-stepping reallocations were only significantly associated with 10 lower BMI, 7 cm lower waist circumference, and 11 lower 2-h plasma glucose. Conclusion Findings suggested that sitting-reduction strategies targeting increased standing, stepping, or both, may benefit cardio-metabolic health. Standing is a simple alternative to sitting, and requires further examination in prospective and intervention studies.