The aim of our prospective study was to quantify phosphate removal during long nocturnal high-flux hemodialysis or hemodiafiltration (HD) with total dialysate collection. Eight patients (two women) were studied for the first-in-the-week HD session that lasted 7–8 h. Total dialysate was collected. Serum and collected dialysate phosphate concentrations were measured every hour. Phosphate removal as assessed from the serum concentration was most important during the first 2 h of HD, and then a plateau was reached. The highest average phosphate concentration in the total dialysate was in the 1st hour, thereafter the concentration decreased but remained stable. The average total removed mass of phosphate quantified from hourly collected dialysate was 5195.7 ± 1898 mg. Phosphate had been removed in a consistent manner during the whole duration of nocturnal HD as assessed through dialysate (despite stable serum phosphate concentration). This could indicate phosphate transfer from intracellular space. The total removed phosphate quantified from the total dialysate collection was higher than previously reported and exceeded the normal phosphate food intake.