© 2022 The Japanese Orthopaedic AssociationBackground: We investigated intramuscular fat (IMF) in quadriceps femoris (QF) and hamstring muscles in the middle-aged women with knee osteoarthritis (KOA). We also examined the relationship between muscular infiltration of QF and hamstring muscles and muscle architecture and physical performance of the women with KOA. Methods: In this cross-sectional study, 72 women were included. Body muscle and fat mass were measured by BIA, isometric muscle strength was evaluated by hand-held dynamometer. IMF and muscle architecture were calculated from rectus femoris (RF), vastus intermedius (VIM), vastus lateralis (VL), vastus medialis (VM), biceps femoris (BF), semitendinosus (ST) and semimembranosus (SM) using B-mode ultrasonography. KOA-related symptoms and functions were assessed with KOOS. The functional performance assessments were evaluated with Stair Climbing Test, 20-Meter Walking Test. Results: Women with KOA had more IMF in RF, VIM, VL, VM and BF, ST, SM muscles compared to the healthy women. Pennation angles decreased as the IMF in the RF, VM, BF and ST decreased. As the IMF of the RF and VM increased isometric knee extensor strength decreased and KOOS symptom score, pain score and ADL score increased in women with KOA. Walking and stair climbing speed deteriorated as the IMF in RF, VIM, VM, BF increased in the middle-aged women. As the IMF in BF increased isometric knee flexor strength decreased and KOOS scores increased. Physical performance scores deteriorated as the IMF in BF increased in middle-aged women with KOA. Conclusion: IMF in QF and hamstring muscles were higher in the middle-aged women with KOA group compared with that in the healthy group. Weakness of the QF and hamstring muscles may due to the changes in architectural properties of muscle depending on muscular infiltration. IMF in knee muscles is an important determining factor in performance and physical function of middle-aged women with KOA.