Bioimpedance analysis (BIA) is a well-known and tested method for body mass and muscular health assessment. Multi-frequency BIA (mfBIA) equipment now makes it possible to assess a particular muscle as a whole, as well as looking at a muscle at the fiber level. The aim of this study was to test the hypothesis that mfBIA can be used to assess the anatomical, physiological, and metabolic state of skeletal muscles. mfBIA measurements focusing on impedance, resistance, reactance, phase angle, center frequency, membrane capacitance, and both extracellular and intracellular resistance were carried out. Eight healthy human control subjects and three selected cases were examined to demonstrate the extent to which this method may be used clinically, and in relation to training in sport. The electrode setup is shown to affect the mfBIA parameters recorded. Our recommendation is the use of noble metal electrodes in connection with a conductance paste to accommodate the typical BIA frequencies, and to facilitate accurate impedance and resistance measurements. The use of mfBIA parameters, often in conjunction with each other, can be used to reveal indications of contralateral muscle loss, extracellular fluid differences, contracted state, and cell transport/metabolic activity, which relate to muscle performance. Our findings indicate that mfBIA provides a noninvasive, easily measurable and very precise momentary assessment of skeletal muscles.