Heterologous protein expression using Pichia pastoris causes metabolic stress on the physiology of host cells, which may compromise the yields of secreted foreign proteins. Thus, understanding these metabolic stresses during secretory expression allows us to circumvent these undesirable effects. We investigated the effect of co-feeding two alternative carbon resources, sorbitol and yeast extract (YE), on the physiology of A3, a P. pastoris strain carrying 18 copies of the porcine insulin precursor (PIP) gene. Comparative transcriptional analysis was performed on 13 selected genes involved in important cellular processes. Results showed that co-feeding of either sorbitol or YE along with methanol improved the performance of A3. The co-feeding of YE enhanced the specific growth rate of A3 and the specific PIP productivity. However, the oxidative stress in the yeast cells increased. The co-feeding of methanol and sorbitol increased the specific growth rate of A3 but did not affect the specific PIP productivity. The transcriptional results suggested that sorbitol may have repressed the expression of foreign proteins. These observations will not only guide the application of mixed feeding strategies but also give a deeper understanding of the metabolic burden in the secretory expression of foreign proteins.