Scale-up of microbial bioprocesses to production scale is accompanied by significant changes in growth conditions. One aspect are rapidly changing substrate concentrations due to prolonged mixing times. The impact of substrate gradients on the physiology of Saccharomyces cerevisiae has been studied for glucose but no other industrially relevant carbohydrate. With different transport mechanism, non-glucose sugars will lead to a different intracellular response. Here we studied the response of S. cerevisiae to gradients for four different sugars: glucose, fructose, sucrose and maltose. To study the impact of the carbon sources as well as the large-scale gradients, steady-state and dynamic feast/famine cultivation conditions were applied. The physiology, intracellular metabolome as well as the proteome were compared. Especially, gradients of maltose lead to a significant decrease in biomass yield. Under dynamic glucose, fructose and sucrose conditions S. cerevisiae was able to maintain the biomass yield of steady-state conditions. Although the physiology was very comparable for these sugars, the intracellular metabolome and proteome changed. The concentration of upper glycolytic enzymes decreased for glucose and maltose (up to -60% and -40% respectively), while an increase was observed for sucrose and fructose when exposed to gradients. At the same time, enzymes of lower glycolysis were increased. Interestingly, common stress-related proteins were decreased during dynamic conditions. The observed adaptations to repeating gradients highlight the importance to study physiology and metabolism under dynamic conditions to obtain results that are relevant for the envisioned large-scale process.