Fatty acid analysis requires standardized collection and storage of samples, which can be a challenge under field conditions. This study describes the effect of storage temperature on fatty acid composition in two sets of whole blood samples collected from 66 children in a rural area in Cambodia. The samples were stored with butylated hydroxytoluene at -20 °C and -80 °C and the latter required extra transfers due to storage facility limitation. Fatty acid composition was analyzed by high-throughput gas-chromatography and evaluated by paired t-tests and Bland-Altman plots. Total amounts of fat in -20 °C and -80 °C samples did not differ, but there was relatively more highly unsaturated fatty acids (15.8 ± 2.7 vs. 14.4 ± 2.5%, p < 0.001) and a lower n-6/n-3 ratio (6.4 ± 1.4 vs. 6.9 ± 1.4, p < 0.001) in the -20 °C samples. Our results indicate that the importance of storage temperature should be evaluated in the context of storage facility availability and risk of temperature fluctuations during transport.