The anadromous sea lamprey (Petromyzon marinus) stop feeding during spawning migration; following the migration effort, the energy reserves of their tissues are expected to be mobilised and depleted. The present work aimed at testing if mobilisation of nutrients occurred in sea lampreys during the 2011 spawning run in the Minho River. Professional fishermen at three sampling sites captured the individuals used: two at the beginning of the freshwater spawning migration (one at the river mouth and one upstream the estuary) and one near the spawning grounds (35 km upstream the estuary limit). The total lipids (Folch extraction), protein (nitrogen conversion), energy content (direct calorimetry) and fatty acids (GC/EI-MS-SIM) were determined in the carcass (n = 19) and female liver (n = 8) and gonads (n = 8). The results indicated that a reduction of carcass dry matter during migration was mainly caused by a lipid decrease (51.0 ± 2.46% downstream and 38.0 ± 2.92% upstream, LSmean ± SE). In females, a decrease in the measured amounts of liver lipids was also observed (4.28 ± 2.637 g downstream and 0.64 ± 2.206 g upstream, LSmean ± SE) and the monounsaturated fatty acids consistently composed the larger fraction of lipids in all tissues (ranging between 42.9 ± 4.20% and 54.9 ± 3.43% of total fatty acids, LSMean ± SE). The results suggest a dynamic nutrient mobilisation and the major importance of liver fatty acids (such as 14:1n-5 and 16:1n-7) as possible energy sources for embryonic development or combustion during spawning migration. The P. marinus individuals captured at the intermediate sampling site (approximately 35 km upstream river mouth) in the middle of the season seemed as ready to spawn as the upstream sea lampreys.