Background: Several methods have been developed to assess the hydration status in chronic hemodialysis (HD) patients. The aim of this study was to compare body bioimpedance spectroscopy (BIS) with ultrasound (US) lung comet score (ULCs), B-type natriuretic peptide (BNP) and inferior vena cava diameter (IVCD) by US for the estimation of dry weight before and after HD and to analyze all methods in terms of fluid status variations induced by HD. An additional aim of this study was to establish the interoperator reproducibility of these methods. Methods: Two nephrologists evaluated BIS, ULCs, IVCD during inspiration (min) and expiration (max), the inferior vena cava collapsibility index (IVCCI) as well as BNP before and after HD in 30 patients. The same operators measured BIS, ULCs and IVCD in 28 HD patients in a blinded fashion. Results: There was a significant reduction in BIS, ULCs, IVCD and BNP after HD (p < 0.001), but a less significant reduction in IVCCI (p = 0.13). There was a significant correlation between BIS and ULCs, BNP and indexed IVCD (IVCDi)min (p < 0.05) before and after HD, and between BIS and IVCDimax only before HD. Conclusion: All methods were able to describe hyperhydration before and after HD, except for IVCCI after HD. All techniques correlated with BIS before HD. After HD, ULCs correlated better with BIS than IVCD in terms of evaluation of fluid status. It could be expected that the ULCs can give a real-time evaluation of interstitial water. The reproducibility of the measurement of BIS, IVCD and ULCs between the two operators was high.