BACKGROUND We quantified the alternate muscle activity among four synergists of the quadriceps femoris (QF), including the vastus intermedius (VI), during low-level sustained contraction. METHODS Surface electromyograms (EMGs) were recorded from the VI, vastus lateralis (VL), vastus medialis (VM), and rectus femoris (RF) in 11 healthy men during isometric knee extension at 2.5% maximum voluntary contraction for 60 min to determine alternate muscle activity among the four synergists of the QF. RESULTS Alternate activity was primarily found between the RF and VI, VL, or VM, and rarely found among the vasti muscles. Multiple muscle comparison revealed the duration of alternate activity in the RF/VI+VL+VM combination remained high throughout the experiment, although the frequency of that combination did not. CONCLUSIONS These results suggested that there is a fixed muscle combination, i.e., RF and the 3 vasti muscles, to perform low-intensity sustained contraction in the human QF.