Objectives. The human skeletal muscle responds immediately under electrical muscle stimulation (EMS), and there is an immediate physiological response in human skeletal muscle. Non-invasive quantitative analysis is at the heart of our understanding of the physiological significance of human muscle changes under EMS. Response muscle areas of human calf muscles under EMS have been detected by frequency difference electrical impedance tomography (fd-EIT). Approach. The experimental protocol consists of four parts: pre-training (pre), training (tra), post-training (post), and relaxation (relax) parts. The relaxation part has three relaxation conditions, which are massage relaxation (MR), cold pack relaxation (CR), and hot pack relaxation (HR). Main results. From the experimental results, conductivity distribution images σ
p
(p means protocol = pre, tra, post, or relax) are clearly reconstructed by fd-EIT as response muscle areas, which are called the M
1 response area (composed of gastrocnemius muscle) and the M
2 response area (composed of the tibialis anterior muscle, extensor digitorum longus muscle, and peroneus longus muscle). A paired samples t-test was conducted to elucidate the statistical significance of spatial-mean conductivities 〈σ
p
〉
M1 and 〈σ
p
〉
M2 in M
1 and M
2 with reference to the conventional extracellular water ratio β
p
by bioelectrical impedance analysis. Significance. From the t-test results, 〈σ
p
〉
M1
and 〈σ
p
〉
M2 have good correlation with β
p
. In the post-training part, 〈σ
post
〉 and β
post
were significantly higher than in the pre-training part (n = 24, p < 0.001). The relax–pre difference ratios of spatial-mean conductivity Δ〈σ
relax–pre
〉 and the relax–pre difference ratios of extracellular water ratio Δβ
relax–pre
in both MR and CR were lower; on the contrary, the Δ〈σ
relax–pre
〉 and Δβ
relax–pre
in HR were significantly higher than those in post–pre difference ratios of spatial-mean conductivity Δ〈σ
post–pre
〉 (n = 8, p < 0.05). The reason for the changes in 〈σ
p
〉
M1 and 〈σ
p
〉
M2 are caused by the changes in muscle extracellular volumes. In conclusion, fd-EIT satisfactorily evaluates the effectiveness of human calf muscles under EMS.