Objective: To compare upper and lower body 5x6s performance between males and females to determine if significant gender differences exist. Methods: Twenty physically active adults (males n=12, females n=8) volunteered to perform an upper and lower body 5x6s. The upper body 5x6s was conducted on a modified electro-magnetically braked cycle ergometer, while the lower body 5x6s was conducted on an electronically braked cycle ergometer using a flywheel braking force corresponding to 5% and 7.5% bodyweight respectively. During the upper body 5x6s, participants were restrained at the waist with an adjustable seatbelt in an attempt to minimise the contribution from the lower body. Body composition was assessed using dual-energy X-ray absorptiometry (DEXA). Data are reported as means ± standard deviation (SD). A level of significance of 5% (P<0.05) was adopted in all analyses. Results: Significant (p<0.001) differences occurred in both absolute (W) and relative (W·kg-1) PP and mean power (MP) between genders during the upper body 5x6s, with differences remaining even when relative to lean body mass (LBM) and active muscle mass (AMM). In contrast, PP and MP for the lower body 5x6s was only significantly different between genders when expressed in absolute (W) and relative (W·kg-1) terms. Conclusion: While both lower body 5x6s performance and lower body 5x6s predictors do not differ significantly between genders, significant differences do exist for upper body 5x6s performance and performance predictors. Even after allometric scaling and training status have been considered, significant gender differences still remain for upper body 5x6s performance, suggesting that unknown intrinsic muscle properties may be responsible for the significant gender differences observed. Thus, females must be trained differently to their male counterparts during upper body exercise.