Lymph collection via insertion of a cannula into a lymphatic vessel is the most commonly used procedure to quantify the transport of pharmaceutical agents into lymph following administration to rodents. Further, lymph is derived from interstitial fluid draining specific organs and tissues such that compositional analysis of lymph collected via cannulation can provide useful information about biochemical and immune cell changes in different patho/physiological states, as well as being a resource for biomarker discovery. Nevertheless, lymph cannulation is a challenging procedure, and continuous collection of lymph for extended periods can lead to lymphocytopenia. Just as important, prolonged lymph collection might deplete other major components in lymph and plasma such as proteins and lipids, yet this has not been reported previously. Therefore, we investigated the effect of thoracic lymph collection in rats on the concentration of protein components (e.g. albumin) and specific lipids (total cholesterol and triglycerides) in lymph and plasma over time for 48 h after lymph cannulation. This study suggests that the level of total protein and albumin, but not lipids, is diminished over time during thoracic lymph collection in rats. We also provide evidence that this depletion impacts the pharmacokinetics of a drug delivery carrier that binds to albumin and lipoproteins. These findings present an important consideration to evaluating the lymphatic transport and pharmacokinetics of pharmaceutical agents that interact with endogenous proteins such as albumin.