Purpose: There is sparse evidence for return to sport criteria after knee injury. Functional performance deficits, particularly in fatigued muscular condition, should be verified prior to the attempt to return to high-risk pivoting sports. The purpose of this study was to generate reference values for the limb symmetry index (LSI) of healthy subjects in fatigued and non-fatigued muscular condition in a newly designed test battery. Methods: Forty-two healthy subjects [22 females, 20 males; mean (SD) age 30.4 (6.6) years] were evaluated using a test battery consisting of an isometric strength test, a series of five single-leg hop tests and an integrated fatigue protocol. Subjective physical activity was assessed with the Tegner Activity Scale (TAS). The cut-off values for healthy subjects were calculated considering the fifth percentile as the minimum reference value for the LSI and single-leg hop distance. Results: The mean (SD) overall LSI was 98.8% (4.6). No significant gender or age specific differences in limb symmetry were observed. The comparison of the non-fatigued LSI with the overall LSI revealed no clinically relevant change due to muscular fatigue. Repeated measures ANOVA revealed a significant within effect on fatigue/non-fatigue condition (F(1,38) = 18.000; p < 0.001, η2 = 0.321) on absolute single-leg hop distance. Moreover, a significant between effect on the TAS-parameter (F(1,38) = 5.928; p = 0.020, η2 = 0.135 between: TAS ≤ 5/TAS > 5) and on gender (F(1,38) = 23.956; p < 0.001, η2 = 0.387) could be detected. Conclusions: The absolute jumping distance in the single-leg hop for distance was significantly reduced due to fatigue. No clinically relevant effect of muscular fatigue was observed on limb symmetry in our study sample. Gender and physical activity are important factors to be considered when interpreting reference values.