To fuel their migratory endurance flights, most birds accumulate large quantities of fat prior to departure. It therefore seems logical that the decision to depart on a migratory flight depends on fuel stores, at least to some extent; very small fuel stores prohibit migratory endurance flight. However, studies linking migrants’ estimated fuel stores (or sometimes actual fat stores) to departure likelihood have provided inconsistent results. Fuel stores are not static though, but highly dynamic. Instead of investigating fuel stores at a given time, we therefore focussed on quantifying the dynamic process of fuelling and how it affects migrants’ motivation to depart. We did so by temporarily caging wild northern wheatears (Oenanthe oenanthe) at a stopover site during autumn migration. Birds were kept under controlled conditions, receiving ad libitum food. Changes in fat mass (measured by quantitative magnetic resonance) and migratory restlessness (an accurate proxy for migrants’ motivation to depart from stopover) were closely monitored. We found that the change in fat mass was a good predictor of the change in migratory restlessness. Individuals accumulating little fat did not show much change in their restlessness, while individuals accumulating much fat showed a strong increase in restlessness. Our study therewith provides clear support for the idea that fat stores affect birds’ motivation to migrate, and shows that for migrating birds, increasing fat stores are an important cue to depart from stopover. Fat accumulation affected the change in migratory restlessness in the same manner for relatively lean and relatively fat birds. Additionally, food intake or changes therein did not explain the change in migratory restlessness we observed. We therefore hypothesize that the fat tissue itself signals changes in energetic state, likely through production and release of hormones, affecting birds’ motivation to migrate.