In this study, we evaluated the effects of experimenter handling, batch effects, and animal-specific variation on proteomic analyses of mucosal scrapings in pigs. We analysed mucosal samples from twelve piglets that were fed two distinct diets. Mucosal samples were obtained from a 60 cm segment collected from the jejunum, which was then divided into six equal segments. Scrapings were collected by two samplersexperimenters, who each handled three of the six segments per pig. Both samplers experimenters were given the exact same instructions for performing the mucosal scrapings. Samples were then randomized and processed in two separate groups on different days to be able to examine batch effects. Protein extraction and digestion was carried out using Midi S-Traps (ProtiFi) and proteins peptides were identified analyzed by using a Fusion Lumos mass spectrometer in Data Independent Acquisition (DIA-MS) mode. Data processing and statistical analysis wasere performed using DIA-NN and RStudio. Our analyses revealed no significant experimenter effects, but we observed small batch effects across datasets and some evidence of biological variation between individual animals. These Our findings emphasize the importance of standardized protocols to minimize experimenter-induced variability and the necessity of correcting for batch effects during data analysis. Biological differences among animals remain are also a crucial factor to be considered in study design, requiring careful consideration when constructingfor experimental designs. This Our study provides a framework for minimising unwanted variability in future proteomic studies, ensuring more reliable and reproducible findings.