A multi-volume 31P-magnetic resonance spectroscopy localization procedure was implemented to compare directly muscle metabolism and proton T2* relaxation properties in the human plantar flexor muscles during exercise. Localized 31P-spectra were collected simultaneously from the medial gastrocnemius, lateral gastrocnemius and soleus muscles during exercise using beta1-insensitive Hadamard Spectroscopic Imaging (HSI). 1H T2*-weighted gradient-echo images were acquired at rest and immediately following high-intensity plantar flexion exercise. T2* mapping of the individual calf muscles showed that plantar flexion with the knee extended produces significant increases (P < 0.0001) in the mean (SEM) T2* of the medial [35.6 (1.2) ms vs 28.5 (0.5) ms at rest] and lateral gastrocnemius [35.6 (0.9) ms vs 26.2 (0.9) ms at rest], but not in the soleus [26.7 (0.6) ms vs 27.3 (0.8) ms at rest]. In accordance with the changes in T2*, the ratio of inorganic phosphate to phosphocreatine (Pi:PCr) and the intracellular muscle pH shifted significantly in the gastrocnemii, while the soleus showed no change in muscle pH and only a moderate increase in Pi-to-Ph. Comparison of spectroscopic and relaxation parameters in both gastrocnemius muscles revealed a significant relationship between post-exercise T2* and intracellular pH (r = 0.72-0.76) and Pi-to-Ph ratios (r = 0.81-0.88) during exercise. Using an improved method of localization, this study confirms the existence of a strong relationship between transverse relaxation properties and the metabolic state in skeletal muscles engaged in heavy exercise.