Lipids play critical roles in modulating membrane protein structure, interactions, and activity. Nanodiscs provide a tunable membrane mimetic that can model these endogenous protein-lipid interactions in a nanoscale lipid bilayer. However, most studies of membrane proteins with nanodiscs use simple synthetic lipids that lack the headgroup and fatty acyl diversity of natural extracts. Prior research has successfully used natural lipid extracts in nanodiscs that more accurately mimic natural environments, but it is not clear how nanodisc assembly may bias the incorporated lipid profiles. Here, we applied lipidomics to investigate how nanodisc assembly conditions affect the profile of natural lipids in nanodiscs. Specifically, we tested the effects of assembly temperature, nanodisc size, and lipidome extract complexity. Globally, our analysis demonstrates that the lipids profiles are largely unaffected by nanodisc assembly conditions. However, a few notable changes emerged within individual lipids and lipid classes, such as a differential incorporation of cardiolipin and phosphatidylglycerol lipids from the E. coli polar lipid extract at different temperatures. Conversely, some classes of brain lipids were affected by nanodisc size at higher temperatures. Collectively, these data enable the application of nanodiscs to study protein-lipid interactions in complex lipid environments.