The potential of eccrine sweat as a bio-fluid of interest for diagnosis and personalized therapy has not yet been fully evaluated, due to the lack of in-depth sweat characterization studies. Thanks to recent developments in the field of omics together with the availability of accredited eccrine sweat collection methods, the analysis of human sweat may now be envisioned as a standardized, non-invasive test for individualized monitoring and personalized medicine. Here, we characterized individual sweat samples, collected from 28 healthy adult volunteers under the most standardized sampling methodology, by applying an optimized Shotgun proteomic analysis. This deep characterization of the sweat proteome allowed the identification of about 1000 unique proteins from which 347 were identified across all samples. Annotation-wise, the study of the sweat proteome unveiled the over-representation of newly addressed Actin dynamics, oxidative stress and proteasome-related functions, in addition to well-described proteolysis and anti-microbial immunity. The sweat proteome composition appeared to be correlated to the inter-individual variability of sweat secretion parameters (water and solute losses). Besides, both gender-exclusive proteins and gender-specific protein abundances were highlighted in spite of the high similarity between human female and male sweat proteomes. In conclusion, standardized sample collection coupled to optimized shotgun proteomics significantly improved the depth of sweat proteome coverage, far beyond previous similar studies. The identified proteins were involved in many diverse biological processes and molecular functions indicating the potential of this bio-fluid as a valuable biological matrix for further studies. Addressing sweat variability, our results prove the proteomic profiling of sweat to be a promising bio-fluid for individualized, non-invasive monitoring and personalized medicine.