AimsIn chronic heart failure (CHF), changes in cardiac function define the course of the disease. The cardiac index (CI) is the most adequate indicator of cardiac function. Interpretation of serial CI measurements, however, requires knowledge of the biological variation of CI. Because measurements of CI can be confounded by the clinical situation or the method applied, biological variation might be subject to the same confounders.Methods and resultsWe prospectively included 50 CHF patients who met rigid criteria for clinical stability. CI was measured by both inert gas rebreathing (IGR) and impedance cardiography (ICG) in weekly intervals over 3 weeks—each measurement performed at rest (IGRrest/ICGrest) and during low‐exercise 10 Watt pedalling (IGR10W/ICG10W). Intra‐class correlation coefficients (ICCs), reference change values, and minimal important differences of CI were determined for IGRrest, ICGrest, IGR10W, and ICG10W. Impedance cardiography and IGR showed moderate agreement at rest (20% (6–36)) and good agreement at 10 Watt (−4% (−23–16)). Depending on time interval, measurement modality for CI, and mode, ICC ranged between 0.42 and 0.78, ICC values for IGR were lower than those for ICG. Reference change value ranged between 3 and 15%, and minimal important difference ranged between 0.2 and 0.5 L/min/m2. Values for IGR were lower at rest and higher at 10 Watt than those for ICG.ConclusionNon‐invasive measurements of CI are stable over time. Measurement modalities for CI, however, are not interchangeable. Biological variation is less pronounced when obtained by ICG. The influence of low‐level exercise on stability of CI depends on the measurement modality.