The slack length of a relaxed skeletal muscle can be reduced by isometric contraction at short lengths ("contract-short conditioning"). This study explored how the effect of contract-short conditioning on muscle slack length is modified by 1) the intensity of the contraction, 2) the delay between the contraction and measurement of slack length, and 3) the amplitude of a stretch delivered to the relaxed muscle after the contraction. Muscle fascicles in the human vastus lateralis muscle were observed with ultrasound imaging while the relaxed muscle was lengthened by flexing the knee. The knee angle at which muscle fascicle slack was taken up was used as a proxy for muscle slack length. Conditioning the muscle with voluntary isometric (fixed-end) contractions at short muscle lengths reduced vastus lateralis muscle slack length, measured 60 s later, by a mean of 10. This effect was independent of contraction intensity from 5% to 100% maximal voluntary contraction. The effect was largest when first observed 5 s after the contraction, decayed about one-third by 60 s, and then remained nearly constant until the last observation 5 min after the contraction. A slow stretch given to the relaxed muscle after contract-short conditioning increased slack length (i.e., reduced the effect of contract-short conditioning). Slack length increased nonlinearly with stretch amplitude. Very large stretches (>30, possibly as large as 90) were required to abolish the effect of contract-short conditioning. The phenomena described here share some characteristics with, and may involve similar mechanisms to, passive force enhancement and muscle thixotropy.NEW & NOTEWORTHY The slack length of a relaxed human skeletal muscle is not fixed; it can be modified by contraction and stretch. Contraction of the human vastus lateralis muscle at short lengths reduces the muscles slack length. Even very weak contractions are sufficient to induce this effect. The effect persists for at least 5 min but can be reduced or abolished with a large-amplitude passive stretch.