Metabolites of human or microbial origin have the potential to be important biomarkers of disease state in cystic fibrosis (CF). Clinical sample collection and storage conditions may impact metabolite abundances with clinical relevance. We measured the change in metabolite composition based on untargeted gas chromatography mass spectrometry (GC-MS) when CF sputum samples were stored at either 4°C, -20°C, or -80°C with one or two freeze-thaw cycles. Daily time points were taken for one week and then weekly for 4 weeks (4°C) and 8 weeks (-20°C). The metabolites in samples stored at -20°C maintained similar abundances compared to -80°C over the course of eight weeks (average change in Bray-Curtis distance: 0.06±0.04), and were also stable after one or two freeze-thaw cycles. However, metabolite profiles of samples stored at 4°C shifted after one day and continued to change over the course of four weeks (average change in Bray-Curtis distance: 0.31±0.12). Several amino acids and other metabolite abundances increased with time when stored at 4°C, but remained constant at -20°C. Storage temperature was a significant factor driving the metabolite composition (PERMANOVA R2 = 0.32 to 0.49, p= <0.001). CF sputum samples stored at -20°C at the time of sampling maintain a relatively stable untargeted GC-MS profile. Samples should be frozen on the day of collection, as more than one day at 4°C impacts the global composition of the metabolites in the sample.