Monitoring fatigue is of vital importance to practitioners, however, logistics and concerns about reliability may impede the use of certain measures. This study aimed to quantify the reliability of potential measures of fatigue; a subjective wellness questionnaire, jump performance tests and tri-axial accelerometer variables derived during sub-maximal shuttle running in elite youth soccer players. A secondary aim was to establish the minimum test duration that could be used for the sub-maximal shuttle run while maintaining good reliability. Seventeen male youth team players (age: 17.4 ± 0.5 years) were assessed on two occasions, spaced seven days apart. Typical error (TE), coefficient of variation (CV%), interclass correlation (ICC) and minimum detectable change (MDC) were calculated for a subjective wellness questionnaire, countermovement jump (CMJ), squat jump (SJ) and drop jump contact time (DJ-CT), jump height (DJ-JH), and reactive strength (DJ-RSI). A novel sub-maximal shuttle running test was also used to assess tri-axial accelerometer data reliability. Results suggest that CMJ, SJ, DJ-CT and DJ-RSI have good test re-test reliability (CV% = 4.5 – 7.7; ICC = 0.80 – 0.88), however DJ-JH did not show acceptable reliability (CV% = 6.0; ICC = 0.76). Good reliability was found for all tri-axial accelerometer variables during a 3 min (2 min analysis) sub-maximal shuttle run (CV% = 2.4 – 8.0; ICC = 0.81 – 0.95), except for % PlayerLoadTM anterior–posterior (%PLAP) (CV% = 7.2; ICC = 0.63). The subjective wellness questionnaire demonstrated poor reliability for all items (CV% = 11.2 – 30.0; ICC = 0.00 – 0.78). The findings from this study provide practitioners with valuable information about the reliability of a range of potential fatigue monitoring measures. This can be used to help make accurate decisions about the magnitude of change in these assessments when used in practice.