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Abstract Objectives The creamatocrit is a simple technique for estimating the lipid content of milk, widely adopted for clinical and research purposes. We evaluated the effect of long-term cryogenic storage on the creamatocrit for human milk. Methods Frozen and thawed milk specimens (n = 18) were subjected to the creamatocrit technique. The specimens were reanalyzed after long-term cryogenic storage (10 years at <70°C). The correlation between pre- and post-storage values was tested, and their differences were analyzed using the Bland–Altman plot. Results The pre- and post-storage values were highly correlated (r = 0.960, p < .0001). The Bland–Altman plot revealed a positive association between their differences and means (Pitman’s test r = 0.743, p < .001), suggesting the presence of nonconstant bias across the creamatocrit range. Long-term storage of human milk may introduce subtle bias to the creamatocrit in replicating pre-storage values. Further research should evaluate whether this bias is statistically correctable.