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Background: High-intensity interval training (HIIT) is a well-established training modality to improve aerobic and anaerobic capacity. However, sex-specific aspects of different HIIT protocols are incompletely understood. This study aimed to compare two HIIT protocols with different recovery periods in moderately trained females and males and to investigate whether sex affects high-intensity running speed and speed decrement.Methods: Fifty moderately trained participants (30 females and 20 males) performed an exercise field test and were randomized by lactate threshold (LT) to one of two time- and workload-matched training groups. Participants performed a 4-week HIIT intervention with two exercise sessions/week: Group 1 (4 × 30,180 HIIT), 30-s all-out runs, 180-s active recovery and Group 2 (4 × 30,30 HIIT), 30-s all-out runs, 30-s active recovery. High-intensity runs were recorded, and speed per running bout, average speed per session, and speed decrement were determined. Blood lactate measurements were performed at baseline and follow-up at rest and immediately post-exercise.Results: Females and males differed in running speed at LT and maximal running speed determined during exercise field test (speed at LT, females: 10.65 ± 0.84 km h−1, males: 12.41 ± 0.98 km h−1, p < 0.0001; maximal speed, females: 14.55 ± 1.05 km h−1, males: 17.41 ± 0.68 km h−1, p < 0.0001). Estimated maximal oxygen uptake was ~52.5 ml kg−1 min−1 for females and 62.6 ml kg−1 min−1 for males (p < 0.0001). Analysis of HIIT protocols revealed an effect of sex on change in speed decrement (baseline vs. follow-up) in that females showed significant improvements only in the 4 × 30:30 HIIT group (p = 0.0038). Moreover, females performing the 4 × 30:30 protocol presented increased speed per bout and average speed per session at follow-up (all p ≤ 0.0204), while no effect was detected for females performing the 4 × 30:180 protocol. Peak blood lactate levels increased in all HIIT groups (all p < 0.05, baseline vs. follow-up), but males performing the 4 × 30:180 protocol showed no difference in lactate levels.Conclusions: If not matched for physical performance, females, but not males, performing a 4 × 30 HIIT protocol with shorter recovery periods (30 s) present increased average high-intensity running speed and reduced speed decrement compared to longer recovery periods (180 s). We conclude that female- and male-specific HIIT protocols should be established since anthropometric and physiological differences across sexes may affect training performance in real-world settings.