During long‐distance migration, many birds experience periods of either prolonged fasting (during endurance flights) or extensive feeding (during stopovers). Despite decades of research on avian metabolism during migration, many questions have remained unanswered, as such research mainly focused on targeted metabolites and fat metabolism. Here, we examined the plasma‐metabolome of two migrating passerine species before they crossed the Sahara Desert. Birds were sampled at two sites populated by Pistacia trees bearing fat‐rich fruits and at an additional site dominated by blooming Eucalyptus trees. The blood samples were analyzed using both GC‐MS and LC‐MS, using an untargeted approach. Examination of metabolic pathways activated during stopovers indicated a crucial role for cycling glucose through the Cori and Cahill cycles in resting and recovery processes. This novel perspective, conducted on free‐ranging birds, suggests the evolution of avian insulin resistance due to factors such as endurance exercise, fasting, and a preference for fatty acid oxidation during migration, akin to cell trauma recovery. We detected significant inter‐site variations in birds' polar and lipophilic metabolic profiles. We interpret the differences in the polar metabolites to be associated with the physiological state of the birds, with birds that are considered to have landed during the night prior to capture showing different metabolic profiles compared to birds that have spent more time at the stopover site. In contrast, distinctions in the lipophilic profiles of birds were associated with variations in the primary food source that was available to them in the different sites. This study underscores the challenge of interpreting commonly used indicators for assessing migrating birds' physiological state, which was predominantly derived from lipid metabolism in complex ecological systems.

Comparative analysis of the plasma metabolome of migrating passerines: novel insights into stopover metabolism