Abstract Background and Aims Long interdialytic interval in thrice-weekly hemodialysis is associated with excess cardiovascular and all-cause mortality risk. Impaired cardiorespiratory fitness is a strong predictor of mortality in hemodialysis. This study investigated differences in cardiorespiratory fitness assessed with cardiopulmonary exercise testing (CPET) between the end of the 2-day and the 3-day interdialytic interval. Method A total of 28 hemodialysis patients, randomized in two different sequences of evaluation underwent CPET and spirometry examination at the end of the 2-day and the 3-day intervals. The primary outcome was the difference in oxygen uptake at peak exercise [VO2peak (ml/kg/min)] assessed with CPET. Volume status was assessed with interdialytic-weight-gain (IDWG), lung ultrasound, bioimpedance spectroscopy (BIS), and inferior vena cava measurements. A total of 14 age- and sex-matched controls were also evaluated. Results Hemodialysis patients at the end of both 2-day and 3-day intervals presented lower values in all major CPET parameters than controls. VO2peak (ml/kg/min) was significantly higher at the end of the 2-day than the 3-day interval (15.24 ± 4.18 vs 13.64 ± 2.76; p < 0.001); similar were the results for VO2peak (ml/min) (1188 ± 257 vs 1074 ± 224; p < 0.001) and VO2peak (%predicted) (58.9 ± 9.2 vs 52.3 ± 8.6; p < 0.001). Numerical but no statistically significant differences were detected in VO2-anaerobic-threshold(ml/kg/min) and VO2-anaerobic-threshold(ml/min) between the two time-points. Maximal work load (90.1 ± 23.2 vs 79.3 ± 25.1; p < 0.001), exercise duration, HR at peak exercise and oxygen pulse also showed lower values at the end of the 3-day interval. FEV1 levels were similar between the two evaluations. Generalized-linear-mixed-model analysis including IDWG as random covariate attenuated the observed differences in VO2peak (ml/kg/min). Changes in BIS-derived overhydration indexes were moderately correlated with changes of VO2peak (ml/kg/min). Conclusion The 3-day interval was associated with further impairment of VO2 at peak exercise. This effect was predominantly driven by excess fluid accumulation during the extra interdialytic day.