BACKGROUND: The precise origin of phosphate that is removed during hemodialysis remains unclear; only a minority comes from the extracellular space. One possibility is that the remaining phosphate originates from the intracellular compartment, but there have been no available data from direct assessment of intracellular phosphate in patients undergoing hemodialysis. METHODS: We used phosphorus magnetic resonance spectroscopy to quantify intracellular inorganic phosphate (Pi), phosphocreatine (PCr), and βATP. In our pilot, single-center, prospective study, 11 patients with ESKD underwent phosphorus ((31)P) magnetic resonance spectroscopy examination during a 4-hour hemodialysis treatment. Spectra were acquired every 152 seconds during the hemodialysis session. The primary outcome was a change in the PCr-Pi ratio during the session. RESULTS: During the first hour of hemodialysis, mean phosphatemia decreased significantly (-41%; P\textless0.001); thereafter, it decreased more slowly until the end of the session. We found a significant increase in the PCr-Pi ratio (+23%; P=0.001) during dialysis, indicating a reduction in intracellular Pi concentration. The PCr-βATP ratio increased significantly (+31%; P=0.001) over a similar time period, indicating a reduction in βATP. The change of the PCr-βATP ratio was significantly correlated to the change of depurated Pi. CONCLUSIONS: Phosphorus magnetic resonance spectroscopy examination of patients with ESKD during hemodialysis treatment confirmed that depurated Pi originates from the intracellular compartment. This finding raises the possibility that excessive dialytic depuration of phosphate might adversely affect the intracellular availability of high-energy phosphates and ultimately, cellular metabolism. Further studies are needed to investigate the relationship between objective and subjective effects of hemodialysis and decreases of intracellular Pi and βATP content. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: Intracellular Phosphate Concentration Evolution During Hemodialysis by MR Spectroscopy (CIPHEMO), NCT03119818.