The aim of the present study was to investigate the effects of multiple repeated sprints on unilateral jump performance and inter-limb asymmetries. Eighteen recreationally active males performed three single leg countermovement jumps (SLCMJ) as baseline data. The repeated sprint protocol was 6 x 40 m with 20 seconds of passive rest between each sprint. This protocol was conducted four times, each set separated by four minutes of rest. Within that rest period, subjects performed one SLCMJ on each limb after two minutes of rest. A one-way ANOVA showed significant reductions (p < 0.05; ES = -0.52 to -0.99) in jump height on both limbs after each set relative to baseline. Inter-limb asymmetries increased at each time point and ranged from 7.62-14.67%, with significant increases in asymmetry seen after sets three (p = 0.046) and four (p = 0.002). Significant increases in sprint time were shown between sprints one and six in each set (p ≤ 0.01). A fatigue index (%) was also calculated and showed an exponential increase from 5.74% (set one) to 13.50% (set four), with significant differences between all sets (p < 0.001) with the exception of sets three and four. Results from this study show that a 6 x 40 m repeated sprint protocol is a sufficient dose for implementing acute fatigue in recreationally active subjects. This was manifested by reductions in jump height at all time points and jump height asymmetries after the third and fourth sets. These findings indicate that jump height from unilateral jump testing may be a useful metric to use during the monitoring process in recreationally trained athletes.