Background Accumulation of middle-weight uraemic toxins in haemodialysis (HD) patients results in increased morbidity and mortality. Whether medium cut-off HD (MCO-HD) improves removal of middle-weight uraemic toxins remains to be demonstrated. Methods This cross-over prospective study included 40 patients randomly assigned to receive either 3months of MCO-HD followed by 3months of high-flux HD (HF-HD), or vice versa. The primary endpoint was myoglobin reduction ratio (RR) after 3months of MCO-HD. Secondary endpoints were the effect of MCO-HD on other middle-weight toxins and protein-bound toxins, and on parameters of nutrition, inflammation, anaemia and oxidative stress. Results Compared with HF-HD, MCO-HD provided higher mean RR of myoglobin (368 versus 57 +/- 13%, P<0.0001), beta2-microglobulin (68 +/- 6 versus 73 +/- 15%, P=0.04), prolactin (32 +/- 13 versus 59 +/- 11%, P<0.0001), fibroblast growth factor 23 (20 +/- 21 versus 41 +/- 22%, P=0.0002), homocysteine (43 +/- 7 versus 46 +/- 9%, P=0.03) and higher median RR of kappa [54 (48-58) versus 70 (63-74)%, P<0.0001] and lambda free light chain (FLC) [15 (9-22) versus 44 (38-49)%, P<0.0001]. Mean +/- SD pre-dialysis levels of beta2-microglobulin (28.4 +/- 5.6 versus 26.9 +/- 5.1mg/L, P=0.01) and oxidized low-density lipoprote (6.9 +/- 4.4 versus 5.5 +/- 2.5pg/mL, P=0.04), and median (interquartile range) kappa FLC [145 (104-203) versus 129 (109-190) mg/L, P<0.03] and lambda FLC [106 (77-132) versus 89 (62-125) mg/L, P=0.002] were significantly lower. Mean albumin levels decreased significantly (38.2 +/- 4.1 versus 36.9 +/- 4.3 g/L, P=0.004), without an effect on nutritional status as suggested by unchanged normalized protein catabolic rate and transthyretin level. Conclusions Compared with HF-HD, MCO-HD provides higher myoglobin and other middle molecules RR and is associated with moderate hypoalbuminemia. The potential benefits of this strategy on long-term clinical outcomes deserve further evaluation.