Background: Glucocorticoids (GCs) in breastmilk follow the maternal hypothalamus– pituitary–adrenal axis activity and may affect the offspring’s growth and neurodevelopment. There is some evidence suggesting that macronutrients in breastmilk also fluctuate throughout the day. We aimed to research whether GCs and macronutrients are correlated in multiple breastmilk samples obtained over a 24-h period. Methods: A total of 10 mothers provided 45 breastmilk samples collected over a 24-h period. Cortisol and cortisone levels were determined by LC–MS/MS, and macronutrients were measured with mid-infrared spectroscopy. Correlations between breastmilk GCs and macronutrients were assessed with Pearson correlations and linear mixed models. Results: No associations were found between breastmilk GCs and macronutrients (cortisol: β-0.1 (95% confidence interval: −1.0 to 0.7), −4.9 (−12.9 to 3.1) for fat, protein, and carbohydrates, respectively; and −0.3 (−5.6 to 5.0) and cortisone: 0.0 (−2.5 to 2.5), −17.4 (−39.8 to 5.0), and −2.7 (−17.7 to 12.3)) for fat, protein, and carbohydrates, respectively. Adjusting for the time of collection to account for GC rhythmicity did not change the results. Conclusion: We found no associations between GCs and macronutrients in human breastmilk. The excretion of GCs in breastmilk and the effects of breastmilk GCs on offspring are, therefore, likely independent of the excretion and effects of the macronutrients.