Purpose: Increasing protein intake during energy restriction (ER) attenuates lean body mass (LBM) loss in trained males. However, whether this relationship exists in trained females is unknown. This study examined the impact of higher compared to lower protein intakes (35% versus 15% of energy intake) on body composition in trained females during 2 weeks of severe ER. Methods: Eighteen well-trained females completed a 1-week energy balanced diet (HD100), followed by a 2-week hypoenergetic (40% ER) diet (HD60). During HD60, participants consumed either a high protein (HP; 35% protein, 15% fat) or lower protein (CON; 15% protein, 35% fat) diet. Body composition, peak power, leg strength, sprint time, and anaerobic endurance were assessed at baseline, pre-HD60, and post-HD60. Results: Absolute protein intake was reduced during HD60 in the CON group (from 1.6 to 0.9 g·d·kgBM −1) and maintained in the HP group (~ 1.7 g·d·kgBM −1). CON and HP groups decreased body mass equally during HD60 (− 1.0 ± 1.1 kg; p = 0.026 and − 1.1 ± 0.7 kg; p = 0.002, respectively) and maintained LBM. There were no interactions between time point and dietary condition on exercise performance. Conclusion: The preservation of LBM during HD60, irrespective of whether absolute protein intake is maintained or reduced, contrasts with findings in trained males. In trained females, the relationship between absolute protein intake and LBM change during ER warrants further investigation. Future recommendations for protein intake during ER should be expressed relative to body mass, not total energy intake, in trained females.