PURPOSE: To explore the complex interface between the tear film, a unique mucosal fluid which is fundamental to ocular homeostasis and optimal vision, and an in-situ contact lens. This study exploits the use of a unique tear envelope (TE) extraction technique, which harvests the material-influenced layer of tear film that is in intimate contact with the lens during wear, to specifically investigate the influence of contact lens wear on tear film protein dynamics. METHODS: TEs were collected from freshly removed worn lens using a novel microcentrifuge 'piggyback' technique. Two distinct ex vivo studies were performed to investigate the key influencing factors involved. Non lens-wearing tear samples were also collected from all wearers. A compositional protein profile for each TE and tear film (TF) sample was obtained using an Agilent 2100 Bioanalyzer lab-on-a-chip microfluidic assay which detected proteins in a 14-230 kDa range. RESULTS: The data demonstrated that the TE protein compositional profile was quite distinct from either that of tear components deposited on the lens or those held in the tear menisci. For example, for one of the participant subgroups the tear protein average values in tears (n = 39) were determined at 35.2 ± 2.5 % lysozyme, 17.2 ± 0.6 % lipocalin, 7.3 ± 1.6 % IgA, 20.3 ± 1.3 % lactoferrin and 0.4 ± 0.4 % albumin as a function of total protein detected. In contrast, the average TE values were measured at 49.2 ± 3.7 %, 21.3 ± 3.9 %, 7.8 ± 1.6 % and 10.2 ± 1.7 % and 1.3 ± 2.8 % respectively with omafilcon A wear. In addition, 63 % of all TE samples (n = 180) (wearing lotrafilcon B and omafilcon A lenses) were albumin positive compared with only 19 % of all pre-lens insertion tear film samples (n = 237). CONCLUSIONS: The TE approach not only allows material differentiation, but it can determine changes in the ocular host response that may otherwise be missed by sole non lens-wearing tear film sample analysis.