Equine motor neuron disease (EMND) is a neurodegenerative disorder similar to the sporadic form of human amyotrophic lateral sclerosis. This study was conducted to quantify myofiber plasticity in response to EMND. Deep M. gluteus medius biopsy samples from eight horses with an ante mortem diagnosis of EMND, which in five cases was later confirmed by post mortem examination of spinal cord and peripheral nerves, were examined by combined methodologies of electrophoresis of myosin heavy chains (MyHC), muscle enzymes and substrate biochemistry, immunohistochemistry of MyHCs and sarcoendoplasmic Ca2+-ATPase (SERCA) isoforms, quantitative histochemistry of succinic dehydrogenase, glycerol-3-phosphate dehydrogenase, periodic acid-Schiff and capillaries, and photometric image analysis. The data were compared with muscle biopsies from healthy controls. Histopathological findings of EMND were observed in muscle biopsy specimens from all cases, but the severity and intra-biopsy extent varied from case to case. Compared with controls, muscle biopsy samples from EMND horses had a lower percentage of MyHC type I fibers, higher percentages of hybrid IIAX and pure IIX fibers, significant atrophy of all muscle fiber types, reduced oxidative capacity, increased glycolytic capacity, diminished intramuscular glycogen, lower capillary-to-fiber ratio, a higher ratio of myofibers expressing SERCA1a to SERCA2a isoforms, and a lower percentage of fibers expressing phospholamban. Objective discrimination of muscle biopsy specimens according to their healthy status (EMND vs controls) was possible on the basis of their muscular characteristics. A coordinated shift from slow to fast muscle characteristics in contractile and metabolic features of muscle fiber types, together with generalized myofiber atrophy, occurs in EMND and the extent of this change seems to be related to the duration of the disease.