OBJECTIVE Modern lifestyles require people to spend prolonged periods of sitting, and public health messages recommend replacing sitting with as much standing as is feasible. The metabolic/energy cost (MEC) of sitting and standing is poorly understood, and MEC associated with a transition from sitting to standing has not been reported. Thus, we carefully quantified the MEC for sitting, standing and sit/stand transitions, adjusting for age and fat-free mass (FFM) in a sample of adults with no known disease. METHODS Participants (N = 50; 25 women), 20–64 years, randomly performed three conditions for 10 min each (sitting, standing, 1 sit/stand transition min(−1) and then sitting back down). MEC was measured by indirect calorimetry and FFM by dual-energy X-ray absorptiometry. RESULTS V̇O2 (ml kg(−1) min(−1)) for sitting (2.93 ± 0.61; 2.87 ± 0.37 in men and women respectively), standing (3.16 ± 0.63; 3.03 ± 0.40), and steady-state cost of repeated sit/stand transitions (1 min(−1)) (3.86 ± 0.75; 3.79 ± 0.57) were significantly different regardless of sex and weight (p < 0.001). EE (kcal min(−1)) also differed from sitting (1.14 ± 0.18; 0.88 ± 0.11), to standing (1.23 ± 0.19; 0.92 ± 0.13), and sit/stand transitions (1 min(−1)) (1.49 ± 0.25; 1.16 ± 0.16). Heart-rate increased from sitting to standing (~13 bpm; p < 0.001). Neither sex nor FFM influenced the results (p ≥ 0.05). CONCLUSIONS This study found in a sample of adults with no known disease that continuous standing raised MEC 0.07 kcal min(−1) above normal sitting. The transition from sitting to standing (and return to sitting) had a metabolic cost of 0.32 kcal min(−1) above sitting. Therefore, public health messages recommending to interrupt sitting frequently should be informed of the modest energetic costs regardless of sex and body composition.