Inhomogeneities in production-scale bioreactors influence microbial growth and product quality due to insufficient mixing and mass transfer. For this reason, lots of efforts are being made to investigate the effects of gradients that impose stress in large-scale reactors in laboratory scale. We have implemented a scale-down model which allows separating a homogeneous part, a stirred tank reactor (STR), and a plug flow reactor (PFR) which mimics the inhomogeneous regimes of the large-scale fermenters. This scale-down model shows solutions to trigger oxygen limited conditions in the PFR part of the scale-down setup for physiological analysis. The goal of the study was to investigate the scale-up relevant physiological responses of Pichia pastoris strain to oxygen limited process conditions in the above mentioned two-compartment bioreactor setup. Experimental results with non-induced cultures show that the specific growth rate significantly decreased with increasing the exposure time to oxygen limitation. In parallel more by-products were produced. Examining physiological scalable key parameters, multivariate data analyses solely using on-line data revealed that different exposures to the oxygen limitation significantly affected the culture performance. This work with the small scale-downs setup reflects new approaches for a valuable process development tool for accelerating strain characterization or for verifying CFD simulations of large-scale bioreactors. As a novel methodological achievement, the combination of the two-compartment scale-down system with the proposed multivariate techniques of solely using online data is a valuable tool for recognition of stress effects on the culture performance for physiological bioprocess scale-up issues. (C) 2013, The Society for Biotechnology, Japan. All rights reserved.