In bovine milk serum, the whey proteins with the highest N-glycan contribution are lactoferrin, IgG, and glycosylation-dependent cellular adhesion molecule 1 (GlyCAM-1); GlyCAM-1 is the dominant N-linked glycoprotein in bovine whey protein products. Whey proteins are base ingredients in a range of food products, including infant formulas. Glycan monosaccharide composition and variation thereof may affect functionality, such as the interaction of glycans with the immune system via recognition receptors. It is therefore highly relevant to understand whether and how the glycosylation of whey proteins (and their functionality) can be modulated. We recently showed that the glycoprofile of GlyCAM-1 varies between cows and during early lactation, whereas the glycoprofile of lactoferrin was highly constant. In the current study, we evaluated intercow differences and the effects of macronutrient supply on the N-linked glycosylation profiles of the major whey proteins in milk samples of Holstein-Friesian cows. Overall, approximately 60% of the N-glycan pool in milk protein was sialylated, or fucosylated, or both; GlyCAM-1 contributed approximately 78% of the total number of glycans in the overall whey protein N-linked glycan pool. The degree of fucosylation ranged from 44.8 to 73.3% between cows, and this variation was mainly attributed to the glycans of GlyCAM-1. Dietary supplementation with fat or protein did not influence the overall milk serum glycoprofile. Postruminal infusion of palm olein, glucose, and essential AA resulted in shifts in the degree of GlyCAM-1 fucosylation within individual cows, ranging in some cases from 50 to 71% difference in degree of fucosylation, regardless of treatment. Overall, these data demonstrate that the glycosylation, and particularly fucosylation, of GlyCAM-1 was variable, although these shifts appear to be related more to individual cow variation than to nutrient supply. To our knowledge, this is the first report of variation in glycosylation of a milk glycoprotein in mature, noncolostral milk. The functional implications of variable GlyCAM-1 fucosylation remain to be investigated.