The human gastrocnemius was examined with and without creatine supplementation under the conditions of rest, ischemic fatigue (IF), and recovery to perturb the pool sizes and equilibrium between phosphocreatine (PCr) and creatine (Cr). ¹H- and ³¹P-magnetic resonance spectroscopy (MRS) were used to examine the total creatine (tCr) pool in each of the metabolic states. ³¹P-MRS monitored the depletion of the PCr peak during IF to <5% of that at rest. ¹H-MRS focused on the tCr methyl peak at 3.02 ppm (dipolar coupled triplet), at which point it was expected that the triplet peak intensity would be similar both in IF and rest. Initial ¹H-MRS data showed the peak intensity during IF decreased, suggesting a change in tCr pool size. Subsequent studies of transverse relaxation time (T₂) revealed that this decline was primarily due to a more rapid T₂ decay of the tCr peak in IF (T₂ ~40 ms) compared with at rest (T₂ ~162 ms). Because Cr is the major contributor to tCr in IF, it is possible that there is a pool of Cr displaying reduced mobility in vivo. Moreover, the residual dipolar coupled triplet observed at rest collapsed into a broad singlet during IF, suggestive of significant changes in the ordered environment experienced at rest for PCr compared with when it is converted to Cr during IF. In addition, these data suggest that in ¹H-MRS studies whose goals include quantitative estimates of tCr pool sizes, standardized metabolic conditions or careful T₂ evaluations will be required.