Establishing the effect of fluctuating extracellular fluid (ECF) volume on muscle strength in people with end‐stage renal disease (ESRD) on hemodialysis (HD) is essential, as inadequate hydration of the skeletal muscles impacts its strength and endurance. Bioelectrical impedance spectroscopy (BIS) has been a widely used method for estimating ECF volume of a limb or calf segment. Magnetic resonance imaging (MRI)‐acquired transverse relaxation times (T2) has also been used for estimating ECF volumes of individual skeletal muscles. The purpose of this study was to determine the association between T2 (gold standard) of tibialis anterior (TA), medial (MG), and lateral gastrocnemius (LG), and soleus muscles and calf BIS ECF, in healthy and in people with ESRD/HD. Calf BIS and MRI measures were collected on two occasions before and after HD session in people with ESRD/HD and on a single occasion for the healthy participants. Linear regression analysis was used to establish the association between these measures. Thirty‐two healthy and 22 participants on HD were recruited. The association between T2 of TA, LG, MG, and soleus muscles and ratio of calf BIS‐acquired ECF and intracellular fluids (ICF) were: TA: β = 0.30, P > 0.05; LG: β = 0.37, P = 0.035; MG: β = 0.43, P = 0.014; soleus: β = 0.60, P < 0.001. For the HD group, calf ECF was significantly associated with T2 of TA (β = 0.44, P = 0.042), and medial gastrocnemius (β = 0.47, P = 0.027) following HD only. Hence BIS‐acquired measures cannot be used to measure ECF volumes of a single muscle in the ESRD/HD population; however, BIS could be utilized to estimate ratio of ECF: ICF in healthy population for the LG, MG, and soleus muscles.