Background Total and high-molecular-weight (HMW) adiponectin have been associated with endocrine and cardiovascular pathology. As no gold standard is available, the discussion about biological relevance of isoforms is complicated. In our study we perform a method comparison between two commercially available assays measuring HMW and total adiponectin, in various patient groups, thus contributing further to this discussion. Methods We determined levels of HMW and total adiponectin using assays by Lumipulse® and Millipore® respectively, in 126 patients with different clinical characteristics (n = 29 healthy volunteers, n = 22 dialysis patients, n = 25 elderly with body mass index (BMI) < 21 kg/m2, n = 25 elderly with BMI 30–35 kg/m2, n = 26 children). Results The Passing & Bablock regression analysis resulted in HMW adiponectinLUMIPULSE ∗ 0.5 − 0.9 = total adiponectinMILLIPORE, albeit with significant deviation from linearity (p < 0.001). Pearson's correlation was R = 0.987 (p = 0.000). No significant differences between patient groups were observed (p = 0.190). Conclusions The HMW/total adiponectin ratio varies with total adiponectin concentration independent of clinical conditions studied. Our results imply that total and HMW adiponectin have similar utility when assessing adiponectin levels in blood, as the ratio is independent of clinical condition.