AbstractIn this study, a previously developed mini‐bioreactor, the Biocurve, was used to identify an informative stimulus–response experiment. The identified stimulus–response experiment was a modest 50% shift‐up in glucose uptake rate (qGLC) that unexpectedly resulted in a disproportionate transient metabolic response. The 50% shift‐up in qGLC in the Biocurve resulted in a near tripling of the online measured oxygen uptake (qO2) and carbon dioxide production (qCO2) rates, suggesting a considerable mobilization of glycogen and trehalose. The 50% shift‐up in qGLC was subsequently studied in detail in a conventional bioreactor (4 l working volume), which confirmed the results obtained with the Biocurve. Especially relevant is the observation that the 50% increase in glucose uptake rate led to a three‐fold increase in glycolytic flux, due to mobilization of storage materials. This explains the unexpected ethanol and acetate secretion after the shift‐up, in spite of the fact that after the shift‐up the qGLC was far less than the critical value. Moreover, these results show that the correct in vivo fluxes in glucose pulse experiments cannot be obtained from the uptake and secretion rates only. Instead, the storage fluxes must also be accurately quantified. Finally, we speculate on the possible role that the transient increase in dissolved CO2 immediately after the 50% shift‐up in qGLC could have played a part in triggering glycogen and trehalose mobilization. Copyright © 2012 John Wiley & Sons, Ltd.