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The extracellular fluid space is the site of intercellular communication and represents an important source of mediators that can shed light on the parenchymal environment. Sampling of this compartment using continuous microdialysis allows assessment of the temporal changes in extracellular mediators involved in tissue homeostasis and disease processes. However, novel biomarker identification is limited by the current need to utilize specific, targeted molecular assays. The aim of our study was to explore the use of qualitative and quantitative proteomic approaches to define the protein content of dermal dialysate. Timed dermal dialysate samples were collected from healthy human volunteers for 5 h following probe insertion, using a 3,000-kDa MWCO membrane perfused at a rate of 3 μl/min. Dialysate proteins were identified using GeLC–MS/MS and iTRAQ approaches and functions assigned according to the Gene Ontology classification system. More than 80 proteins (size range 11–516 kDa) originating from both extracellular and intracellular fluid space were identified using the qualitative approach of GeLC–MS/MS. Quantitative iTRAQ data were obtained for 27 proteins with relative change ratios between consecutive timed samples showing changes of >1.5-fold. Interstitial proteins can be identified and measured using shotgun proteomic techniques and changes detected during the acute inflammatory response. Our findings provide a platform from which to explore novel protein biomarkers and their modulation in health and disease.