Facioscapulohumeral muscular dystrophy (FSHD) is an untreatable disease, characterized by asymmetric progressive weakness of skeletal muscle with fatty infiltration. Although the main genetic defect has been uncovered, the downstream mechanisms causing FSHD are not understood. The objective of this study was to determine natural disease state and progression in muscles of FSHD patients and to establish diagnostic biomarkers by quantitative MRI of fat infiltration and phosphorylated metabolites. MRI was performed at 3T with dedicated coils on legs of 41 patients (28 men/13 women, age 34-76 years), of which eleven were re-examined after four months of usual care. Muscular fat fraction was determined with multi spin-echo and T1 weighted MRI, edema by TIRM and phosphorylated metabolites by 3D (31)P MR spectroscopic imaging. Fat fractions were compared to clinical severity, muscle force, age, edema and phosphocreatine (PCr)/ATP. Longitudinal intramuscular fat fraction variation was analyzed by linear regression. Increased intramuscular fat correlated with age (p<0.05), FSHD severity score (p<0.0001), inversely with muscle strength (p<0.0001), and also occurred sub-clinically. Muscles were nearly dichotomously divided in those with high and with low fat fraction, with only 13% having an intermediate fat fraction. The intramuscular fat fraction along the muscle's length, increased from proximal to distal. This fat gradient was the steepest for intermediate fat infiltrated muscles (0.07±0.01/cm, p<0.001). Leg muscles in this intermediate phase showed a decreased PCr/ATP (p<0.05) and the fastest increase in fatty infiltration over time (0.18±0.15/year, p<0.001), which correlated with initial edema (p<0.01), if present. Thus, in the MR assessment of fat infiltration as biomarker for diseased muscles, the intramuscular fat distribution needs to be taken into account. Our results indicate that healthy individual leg muscles become diseased by entering a progressive phase with distal fat infiltration and altered energy metabolite levels. Fat replacement then relatively rapidly spreads over the whole muscle.