Road pavements are exposed to traffic and temperature effects that produce distress, mainly cracking that leads to pavement failure, after which rehabilitation actions are necessary to extend pavement life. Bio-rejuvenators have been used to recover the bitumen properties lost due to fatigue of the asphalt mixtures and ageing due to temperature variations and oxidation. Thus, this paper investigates the healing of bitumen modified with bio-oil derived from biodiesel production. One base bitumen in three different ageing stages (unaged, short and long-term ageing) was used for adding up to 3% of bio-oil to the bitumen. In addition to consistency and rheological tests to characterise the modified bitumen, fatigue resistance tests with and without rest periods were performed to assess the healing through three indexes, relating the complex shear modulus, the number of cycles to failure and the amount of damage, after a rest period. It was verified that the healing follows an exponential law with the rest period, but the number of cycles in each loading period decreases compared to the previous period. For a 15-minute rest period, the healing was limited to 26.9%, but the bio-oil contributed significantly to that restoration.