Knee osteoarthritis (KOA) is one of the most prevalent joint disease worldwide. Musculoskeletal modeling (MSM) provides a useful tool to investigate knee joint biomechanics and potential development and progression of KOA. However, for clinical usage, it is crucial to understand how patient-specific properties affect MSM outcomes. This study investigated how uncertainty in the tendon slack length (TSL) of lower limb muscles affect knee joint reaction forces (JRFs) in gait analysis. Two sensitivity analyses were performed, one approach used reported TSL variation in literature and the other approach used a percentage TSL variation. Anatomical variation was investigated through a literature review. The JRF estimations of both approaches together resulted in a maximum variation of 0.27 x BW in AP direction, 2 x BW in SI direction and 0.13 x BW in ML direction. Maximum JRF estimations resulting from reported TSL variation were larger in the loading response in all direction. Maximum JRF estimations in ML direction were resulting from percentage variation. TSL of muscles that were found to be most influential in JRF estimations were medial gastrocnemius, rectus femoris, vastus lateralis and psoas. This research confirmed that within patient-specific MSM for clinical purposes, uncertainty in TSL should be limited. Future research should aim to investigate how this influences AC degeneration for KOA.