Fatty acid levels in milk vary between day and night milking. Many dairy cows are still kept under white light-emitting diode (W-LED) illumination throughout the night, although it is known to disrupt endogenous circadian rhythms. We investigated the effects of whole-night W-LED illumination (125 lux) on milk yield and circadian composition, compared to a natural light–dark (LD) cycle of 10 h light. Mid–late lactation cows (n = 34) that were exposed to natural LD cycle showed circadian variation in milk fat composition, characterized by higher health-promoting monounsaturated fatty acid (MUFA; 24.2 ± 0.4 vs. 23.2 ± 0.4 g/100 g fat, p < 0.001) and lower saturated fatty acid levels (71.2 ± 0.4 vs. 72.5 ± 0.4, p < 0.001) at 13:30 h (day milk) than at 03:30 h (night milk). Compared to natural LD (n = 16), W-LED (n = 18) did not affect milk production or milk fat yields, yet abolished the milking time variation in milk fat composition towards a less healthy fatty acid profile. This lowered MUFA levels of day milk (23.8 ± 0.4 vs. 26.7 ± 0.4, p < 0.01). Therefore, W-LED has no commercial advantage over the tested natural LD cycle, and conversely, even shows circadian disruption. Accordingly, a natural LD cycle of 10 h light is preferable over W-LED from the perspective of cost savings, the cows’ well-being, and preserving the natural milk fat profile, as the nutritional value of the day milk is slightly higher.
