Foot dorsi and plantar flexion affects the pennation angle of skeletal muscle fibers and changes the fiber direction with respect to the main magnetic field, thereby affectingMR spectrum of the muscle. In order to analyze the effect that foot flexion has on the MR spectrum, tibialis anterior (TA) and soleus muscles were studied in humans and rats. Localized MRS was performed at different foot angles in clinical and preclinical settings using a 3 T MRI/MRS GE Excite HD and 7 T Bruker Clinscan scanner, respectively. In this study we show the effect of foot angle variation on total Creatine (tCr) resonance of 1H spectrum at 3.03 and 3.93 ppm for TA and soleus muscles. In addition to this, we observe a 4-line splitting pattern for methylene resonance of tCr in the rat TA spectrum for a specific foot angle. This observation is attributed to the individual splitting of creatine and phosphocreatine of the tCr signal. Novel hydrogel application is demonstrated and used to support our in vivo observations and for the first time splitting of individual resonances of Cr and PCr has been shown in an in vitro set-up.