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Introduction: Hemodialysis (HD) with medium cut‐off (MCO) dialyzers may expand molecular clearance, predominantly larger middle molecules (molecular weight 25–60 kDa). However, the impact of MCO dialyzers on long‐term clearance of various other components of the uremic milieu is unknown. The tRial Evaluating Mid cut‐Off Value membrane clearance of Albumin and Light chains in HemoDialysis patients (REMOVAL‐HD) provided an opportunity to assess the effect of MCO dialyzers on protein‐bound uremic toxins and novel markers of mineral metabolism. Methods: This exploratory sub‐study of REMOVAL‐HD evaluated changes in protein‐bound solutes (total and free indoxyl sulfate [IS] and p‐cresyl sulfate [PCS]) and mineral metabolism markers (intact fibroblast growth factor‐23 [iFGF23], fetuin‐A and endogenous calciprotein particles [CPP‐1 and CPP‐2]). Mid‐week, pre‐HD serum samples were collected at baseline and after 12 and 24 weeks of MCO use in stable adult patients. Change from baseline to Week 12 and 24 was estimated using linear mixed effects models. Findings: Eighty‐nine participants were studied (mean age 67 ± 15 years, 38% female, 51% diabetic, median urine output 200 ml/24 h). Serum iFGF23 was reduced at Week 12 compared to baseline (−26.8% [95%CI −39.7, −11.1], p = 0.001), which was sustained at Week 24 (−21.7% [95%CI ‐35.7, −4.5], p = 0.012). There was no significant change in serum IS, PCS, fetuin‐A, CPP‐1, or CPP‐2. Discussion: The use of a MCO dialyzer over 24 weeks was associated with a sustained reduction in FGF23, while other measured components of the uremic milieu were not significantly altered. Further studies are required to determine whether FGF23 reduction is associated with improved patient outcomes. [ABSTRACT FROM AUTHOR]