The aim of this study was to examine the relationship of a range of in-vivo whole muscle characteristics to determinants of endurance performance. Eleven healthy males completed a cycle ergometer step test to exhaustion for the determination of the lactate threshold, gross mechanical efficiency, peak power and VO₂max. On two separate occasions, contractile and fatigue characteristics of the quadriceps femoris were collected using a specially designed isometric strength-testing chair. Muscle fatigue was then assessed by stimulating the muscle for 3 minutes. Force, rate of force development and rates of relaxation were calculated at the beginning and end of the 3 minute protocol and examined for reliability and in relation to lactate threshold, VO₂max, gross mechanical efficiency and peak power. Muscle characteristics, rate of force development and relaxation rate were demonstrated to be reliable measures. Force drop off over the 3 minutes (fatigue index) was related to lactate threshold (r = -0.72 p ¼ 0.01) but not to VO₂max. The rate of force development related to the peak power at the end of the cycle ergometer test (r = -0.75 p ¼ 0.01). Rates of relaxation did not relate to any of the performance markers. We found in-vivo whole muscle characteristics, such as the fatigue index and rate of force development, relate to specific markers of peripheral, but not to central, fitness components. Our investigation suggests that muscle characteristics assessed in this way is reliable and could be feasibly utilised to further our understanding of the peripheral factors underpinning performance.