Root exudates are plant-derived, exported metabolites that likely shape root-associated microbial communities by acting as nutrients and signals. However, root exudation dynamics are unclear and thus also, if changes in exudation are reflected in changes in microbiome structure. Here, we assess commonalities and differences between exudates of different plant species, diurnal exudation dynamics, as well as the accompanying methodological aspects of exudate sampling. We find that exudates should be collected for hours rather than days as many metabolite abundances saturate over time, that growth in sterile, nonsterile, or sugar-supplemented environments significantly alters exudate profiles (sugar-supplemented: more carbohydrates, less organic acids, organoheterocyclic compounds). Brachypodium distachyon grown in. nonsterile conditions further display distinct root morphology compared to sterile or sugar-supplemented conditions. A comparison of Arabidopsis thaliana, Brachypodium distachyon, and Medicago truncatula shoot, root, and exudate metabolite profiles revealed clear differences between these species, but also a core metabolome for tissues and exudates. Exudate profiles also exhibit a diurnal signature. These findings add to the methodological and conceptual groundwork for future exudate studies to improve understanding of plant-microbe interactions.