Mitochondrial dysfunction is a proposed pathologic mechanism in amyotrophic lateral sclerosis (ALS). Creatine has been shown to stabilize the mitochondrial transition pore, buffer intracellular energy stores, stimulate synaptic glutamate uptake, and scavenge reactive oxygen species. It is suggested that creatine is neuroprotective in vivo and that this effect is dose-dependant. Creatine is safe and well-tolerated but failed to demonstrate efficacy in ALS at 5 and 10 g/day. Experience with creatine in HD patients suggests that creatine may be neuroprotective at higher dosages. The purpose of this study is to establish steady-state serum pharmacokinetics (PK) of creatine at several dosages, and to evaluate the effects of creatine on brain metabolites using proton magnetic resonance spectroscopy ( 1 H-MRS)Six participants with ALS received creatine at three weekly escalating oral dosages (5, 10, and 15 g BID). Creatine blood levels were collected over 5 hours at each visit. MRS was performed at baseline and 5 days after each dose increase. Pharmacokinetic parameters were estimated by measuring the area under the curve (AUC) for each dosage group. Changes in brain metabolite levels on MRS were compared within subjects before and after treatments using repeated measures RM-ANOVA.Mean pre-dose steady-state creatine plasma concentrations were 20.3, 39.3, and 61.5 μg/mL at 5, 10, 15 g BID, respectively. Creatine spectra increased by 8% (p = 0.06) and glutamate + glutamine spectra decreased by 17% (p = 0.039) at higher dosages. There were no safety concerns at any of the dosages.Creatine plasma concentrations increased in a dose-dependant manner. Creatine appears to cross the blood–brain barrier, and oral administration of 15 g BID is associated with increased in vivo brain creatine concentrations and decreased glutamate concentrations. The decrease in brain glutamate concentration with increased creatine dosages is of particular interest given the role of glutamate-mediated injury to motor neurons in ALS. The results of this study support that 15 g BID creatine could be tested as a therapeutic dosage in ALS clinical trials. These data also support MRS as a potential biomarker for creatine effect on brain metabolites.