BACKGROUND: Wearing high heel shoes is thought to increase the risk of knee osteoarthritis in females by altering knee joint alignment and increasing muscle activation. However, direct measurements of knee joint space width using ultrasound have been limited. This study aimed to investigate the effects of 8-cm high-heel shoes on knee joint articular space in healthy young females during standing. METHODS: A cross-sectional study was conducted with 30 healthy females (age 22 ± 1.3 years). Participants' knee medial, lateral and patellofemoral joint space width were measured using an ultrasound imaging device in both barefoot and high-heel shoes conditions. A general linear model was used to compare the impact of high-heel shoes on knee joint articular space with adjusted covariates of demographic and lower limb physical measurement data. RESULTS: Significant reductions in lateral joint space width were observed in both the dominant and non-dominant knees when wearing high-heel shoes compared with barefoot (mean difference: dominant side = 1.63 ± 3.91 mm, non-dominant side = 2.34 ± 2.65 mm, P = 0.008). No significant changes were detected in medial and patellofemoral joint space width. CONCLUSION: Wearing 8-cm high-heel shoes significantly reduces the lateral joint space width of the knee in healthy young females during standing, suggesting increased lateral knee joint stress. The current study underscores the importance of including standing as a test component when measuring joint space changes in future studies and clinical practice to comprehensively evaluate the effects of high-heel usage on knee joint biomechanics.