The objective of this study was to investigate different methods of estimating muscle inactivation, derived from single and multiple voluntary contractions. Ten subjects performed maximal and submaximal leg extensor contractions to determine an interpolation (IT) or central activation ratio (CAR). A superimposed evoked force was compared with the force output of either a voluntary (CAR) or resting evoked contraction (IT ratio), or the ratios were inserted into regression equations (linear, polynomial, exponential). Linear-regression estimates of CAR using doublets and tetanus provided physiologically inaccurate values. Whereas IT ratios using doublets (IT-doublet) and tetanus (IT-tetanus) had a significant difference in only one interaction, IT-tetanus and CAR using a tetanus (CAR-tetanus) estimates provided the most extensive correlation within and between measures. Thus, tetanic stimulation superimposed upon single maximal or multiple contractions seems to provide the most valid measure of muscle inactivation when using the interpolated-twitch technique.