Proteomic studies of bovine milk frequently analyze daily milkings as one pooled sample thus not accounting for possible diurnal fluctuations that may be occurring in the mammary gland. Daily variation in the high abundance proteins (caseins, lactalbumin and lactoglobulin) has been reported, yet little investigation of the impact of diurnal rhythm on the rest of the milk proteins (the low abundance proteins; LAP) has been done. This study investigates diurnal variation of the skim milk LAP profile. The 5-d trial included 3 d used for collection. Milk samples were collected at both AM and PM milkings (0400 and 1500 h) from mid to late lactation multiparous Holsteins (n = 3; DIM = 219.3 ± 77.7). Cows were all housed the same facility, managed under the same protocol, and ad libitum fed the same TMR. Milk samples were flash frozen and stored at −80°C until analysis. Samples were pooled according to time of collection across the week within cow before the addition of a protease inhibitor cocktail and skimmed by repeated centrifugation (4,000 × g). The LAP were isolated through CaCl₂ precipitation of the high abundance proteins followed by ultracentrifugation (189,000 × g for 60 min). The supernatant was lyophilized and reconstituted in PBS and samples were labeled using Tandem Mass Tag (TMT). Protein abundances were determined using LC-MS/MS and PROC MIXED of SAS was used to compare protein abundances in AM versus PM milk samples. A total of 262 proteins were identified using SEQUEST and Mascot within Proteome Discoverer 2.2. Of those proteins, 3 differed in abundance between AM and PM milk samples. Alpha-1-antiproteinase, isocitrate dehydrogenase [NADP], and keratin: type II cytoskeletal 59 kDa, component IV all decreased in abundance from AM to PM milking. The affected proteins are diverse and not comprised in a single gene ontological classification. Results from this trial offers insight into mammary gland changes that may not be accounted for having a single pooled sample to represent a 24-h period that possibly should be considered for future biomarker research. However, these results also suggest that the low abundance skim milk proteome is not significantly affected by time of day.