Gulf War illness (GWI) afflicts military personnel who served during the Persian Gulf War and is notable for cognitive deficits, depression, muscle pain, weakness, intolerance to exercise, and fatigue. Suspect causal agents include the chemicals pyridostigmine (PB), permetrim (PM) and N,N-diethyl-m-toluamide (DEET) used as protectants against insects and nerve gases. No pre-clinical studies have explored the effects on skeletal muscle (SkM). Young male rats were provided PB, PM and DEET at equivalent human doses and physical restraint (to induce stress) for 3 weeks followed a 3-week recovery. GWI gastrocnemius weight was ~ 35% lower versus controls, which correlated with decreases in myofiber area, limb strength, and treadmill time/distance. In GWI rats, SkM fiber type relative abundance changed towards slow type I. Muscle wasting pathway proteins were upregulated while those that promote growth decreased as did mitochondrial endpoints and muscle ATP levels. Proteomic analysis of SkM also documented unique alterations in mitochondrial and metabolic pathways. Thus, exposure to GWI chemicals/stress adversely impacts key metabolic pathways leading to muscle atrophy and loss of function. These changes may account for GWI Veterans symptoms.