Microwave heating of foods may alter their content. Yet, parents commonly heat infant formula in the microwave oven. The study aimed at understanding whether microwave heating of formula changes its fatty acid (FA) composition. Portions of infant formula were prepared and divided in three parts: control (sampled twice: at the start and after 30 minutes), microwave (sampled twice: after reaching 37°C and 50°C), and water bath (sampled twice: after reaching 37°C and 50°C). In thus obtained samples, a total profile of 25 FA was assessed using gas chromatography-mass spectrometry. Overall, fourteen portions were prepared, which were sampled 84 times yielding 2075 individual total FA level measurements. Few differences were identified between the microwave, control, and water bath groups. Microwave warming to 37°C was associated with increases of C12 (median increased by +0.40%, p = 0.0063), C14 (+0.05%, p = 0.0091), and C4 content (+6.90%, p = 0.0131). Microwaving up to 50°C slightly decreased C16:1trans (-5.00%, p = 0.0463) and C18:2trans1 (-5.13%, p = 0.0231). A paired comparison of pooled control and microwaved samples revealed increases in C12 (+0.18%, p = 0.0490) as well as a loss of C18:2trans1 (also -5.13%, p = 0.0073) and C18:2trans3 (-5.56%, p = 0.0042) after microwave treatment. C18:2trans1 (-2.63%, p = 0.0132) and C18:3trans1 (-2.26%, p = 0.0434) were lower in microwaved samples compared with the water bath. A slightly lower C18:2 content was found in the water bath samples than in the control groups (-0.11%, p = 0.0430). None of these differences would remain significant after a correction for multiple comparisons. Microwave heating of infant formula to 37°C or 50°C might marginally alter its total FA profile. Further studies are required to determine whether it alters the rate of free radical formation.