The early quantitative characterization of possible production hosts is a key element in the development of knowledge based biotechnological processes. Despite of scale independent parameters like substrate uptake rates, production rates, maximum growth rates and yields, the performance on mixed substrates, as they occur for example during induction of recombinant protein production, is of great interest. In the present work, pulse and shift experiments with glycerol, glucose, malate, pyruvate and acetate were performed in chemostat cultures of the methylotrophic yeast Pichia pastoris. This allowed the quantitative examination of mixed substrate dynamics and the adaptation to higher growth rates. Different on- and off-line methods were used for a quantitative monitoring of the fermentation process and as input variables to mass balances. Elemental balances in combination with a statistical test for validation of the results were used for data reconciliation. Based on the results, the author discusses the pulsed substances towards their repressive, additive or competitive behaviour as mixed substrates and their effect on the yeasts metabolism.