An acute stress response is a complex process that activates the neuroendocrine and metabolic systems for homeostasis. A study on acute stress is important to understand how an organism adapts to stress for survival. However, most studies have focused on chronic stress, and there are few studies on acute stress. ,ey have analyzed the metabolic alterations in the brain at a particular time after acute stress. ,is study explored the temporal variations of the brain metabolites in the hippocampus after acute restraint stress using proton magnetic resonance spectroscopy. All mice in the acute stress group were physically restrained for two hours in a 50 mL conical tube. A 9.4 Tanimal MRI and MRS scanner was used with point-resolved spectroscopy technique for data acquisition, which was repeated four times without interscan interval. Metabolites were quantified from the data using LCModel with a simulated basis set. Based on the change in concentration of metabolites, the data were statistically analyzed using two-way repeated-measures analysis of variance between groups and a support vector machine for all time points and Student’s ttest with FDR correction for each time point. ,e present study found that the differences between groups are significantly (P < 0.05) presented in alanine and glutamate. ,e effect of time of the two metabolites significantly exists (P < 0.05): the first, second, and third time points in alanine and the first and second time points in glutamate. A combination of stress-specific metabolites (alanine, glutamate, N-acetyl-aspartate) that best reflect the influence of acute stress was determined using a support vector machine. ,ese findings may indicate the importance of the timing of analysis after acute stress and provide new insights into a deeper understanding of acute stress response, including the molecular mechanism of stress-related disorders and stress resilience or vulnerability.