It is important from a techno-economic perspective to understand the challenges associated with developing scalable processes to extract functional proteins from rapeseed cake for applications as functional food ingredients. One of these challenges is to develop a better understanding of the link between upstream mechanical processing of the rapeseed cake and the yield of extracted protein. An initial trial with cold pressed rapeseed cake (CPR) indicated that wet milling had no impact on protein yield. Measurement of protein yields with the Bradford assay indicated that 14% would be recovered. The total protein recovered (5.4% by Kjeldahl analysis) contained a glucosinolate and phytate content that was similar to the rapeseed cake at the start of the process. The fate of the protein extraction was followed during a pilot scale trial with hot-pressed rapeseed cake (HPR), using only stirring followed by sieving, revealing an increase in protein yields, as determined by the Bradford assay, from 8.0% at the start to 22.6% at the end of processing. In addition, only 16% of HPR was solubilized. A similar pilot scale trial with HPR using wet milling and stirring followed by decanting also showed an increase in protein concentration from 15.3% at the start to 23.7% at the end of processing. In addition, 38% of HPR was solubilized. This study provides an insight into what the effect of upstream mechanical processing (wet milling) has in releasing protein from rapeseed cake in order to improve process efficiency. This could have important consequences for upscaling and commercial extraction of rapeseed cake protein.