Dermal interstitial fluid (ISF) shows promise for novel skin diagnostics and as an alternative to invasive blood tests. This dataset includes ISF sampled via a microneedle device from two ex vivo human abdominal skin specimens and the forearms of ten healthy volunteers. Paired blood samples via finger prick were also collected from five volunteers. We studied the ISF proteome in relation to collection time and pressure ex vivo. We also created a reference ISF proteome from the healthy volunteers, compared it to the plasma proteome, and quantified errors from blood contamination.