End-stage kidney disease patients treated with conventional hemodialysis (CHD) are known to have impaired physical performance and protein-energy wasting (PEW). Nocturnal hemodialysis (NHD) was shown to improve clinical outcomes, but the evidence is limited on physical performance and PEW. We investigate whether NHD improves physical performance and PEW. This prospective, multicenter, non-randomized cohort study compared patients who changed from CHD (2-4 times/week 3-5 h) to NHD (2-3 times/week 7-8 h), with patients who continued CHD. The primary outcome was physical performance at 3, 6 and 12 months, assessed with the short physical performance battery (SPPB). Secondary outcomes were a 6-minute walk test (6MWT), physical activity monitor, handgrip muscle strength, KDQOL-SF physical component score (PCS) and LAPAQ physical activity questionnaire. PEW was assessed with a dietary record, dual-energy X-ray absorptiometry, bioelectrical impedance spectroscopy and subjective global assessment (SGA). Linear mixed models were used to analyze the differences between groups. This study included 33 patients on CHD and 32 who converted to NHD (mean age 55 ± 15.3). No significant difference was found in the SPPB after 1-year of NHD compared to CHD (+0.24, [95% confidence interval -0.51 to 0.99], p = 0.53). Scores of 6MWT, PCS and SGA improved (+54.3 [95%CI 7.78 to 100.8], p = 0.02; +5.61 [-0.51 to 10.7], p = 0.03; +0.71 [0.36 to 1.05], p < 0.001; resp.) in NHD patients, no changes were found in other parameters. We conclude that NHD patients did not experience an improved SPPB score compared to CHD patients; they did obtain an improved walking distance and self-reported PCS as well as SGA after 1-year of NHD, which might be related to the younger age of these patients.