Introduction: Training with low-carbohydrate (CHO) availability enhances markers of aerobic adaptation and has become popular to periodize throughout an endurance-training program. However, exercise-induced amino acid oxidation is increased with low muscle glycogen, which may limit substrate availability for postexercise protein synthesis. We aimed to determine the impact of training with low-CHO availability on estimates of dietary protein requirements. Methods: Eight endurance-trained males (27 ± 4 yr, 75 ± 10 kg, 67 ± 10 mL·kg body mass−1·min−1) completed two trials matched for energy and macronutrient composition but with differing CHO periodization. In the low-CHO availability trial (LOW), participants consumed 7.8 g CHO−1·kg−1 before evening high-intensity interval training (10 × 5 min at 10-km race pace, 1 min rest) and subsequently withheld CHO postexercise (0.2 g·kg−1). In the high-CHO availability trial (HIGH), participants consumed 3 g CHO·kg−1 during the day before high-intensity interval training, and consumed 5 g CHO·kg−1 that evening to promote muscle glycogen resynthesis. A 10-km run (~80% HRmax) was performed the following morning, fasted (LOW) or 1 h after consuming 1.2 g CHO·kg−1 (HIGH). Whole-body phenylalanine flux and oxidation were determined over 8 h of recovery via oral [13C]phenylalanine ingestion, according to standard indicator amino acid oxidation methodology, while consuming sufficient energy, 7.8 g CHO·kg−1·d−1, and suboptimal protein (0.93 g·kg−1·d−1). Results: Fat oxidation (indirect calorimetry) during the 10-km run was higher in LOW compared with HIGH (0.99 ± 0.35 g·min−1 vs 0.60 ± 0.26 g·min−1, P < 0.05). phenylalanine flux during recovery was not different between trials (P > 0.05) whereas phenylalanine oxidation (reciprocal of protein synthesis) was higher in LOW compared with HIGH (8.8 ± 2.7 μmol·kg−1·h−1 vs 7.9 ± 2.4 μmol·kg−1·h−1, P < 0.05), suggesting a greater amino acid requirement to support rates of whole-body protein synthesis. Conclusions: Our findings suggest that performing endurance exercise with low-CHO availability increases protein requirements of endurance athletes.