Diverse spectroscopic methods operating at radio frequency depend on a reliable calibration to compensate for the frequency dependent damping of the transmission lines. Calibration may be impeded by the existence of a sensitive interdependence of two or more experimental parameters. Here, we show by combined scanning tunnelling microscopy measurements and numerical simulations how a frequency-dependent conductance response is affected by different DC conductance behaviours of the tunnel junction. Distinct and well-defined DC-conductance behaviour is provided by our experimental model systems, which include C60 molecules on Au(111), exhibiting electronic configurations distinct from the well-known dim and bright C60’s reported so far. We investigate specific combinations of experimental parameters. Variations of the modulation amplitude as small as only a few percent may result in systematic conductance deviations as large as one order of magnitude. We provide practical guidelines for calibrating respective measurements, which are relevant to RF spectroscopic measurements.