The lipid fraction of human milk provides the infant with the fatty acids that are necessary for optimal growth and development. The aim of this study was to investigate the fatty acid composition of human milk at three time points during lactation and its change over time using appropriate statistical methods. Human milk samples from breastfeeding mothers at 6 weeks (n = 706), 6 months (n = 483), and 12 months (n = 81 with all three time points) were analyzed. Centered log-ratio (clr) transformation was applied to the fatty acid data. Principal component analysis (PCA) and generalized linear model-based repeated measure analysis were used to assess changes over time. The total lipid content was significantly higher at 6 months (β = 0.199, p < 0.029) and 12 months of lactation (β = 0.421, p < 0.001). The constituents of C20:3n-6 and C20:3n-3 were lower at 6 months (p < 0.001). Four distinct sub-compositional fatty acid groups were only identified at 12 months of lactation. The inclusion of small fatty acids of small constituent size in the analysis resulted in a shift in the balance between fatty acid constituents. Human milk fatty acid composition during prolonged lactation is different from that of human milk during a short duration of lactation. Our findings support the hypothesis that a combination of multiple fatty acids is important in fatty acid profiling beyond the presentation of individual fatty acids. Furthermore, the high variability of small fatty acids warrants attention because a compositional analysis may show more pronounced changes.