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This study investigated the acute effects of including 30‐second sprints during prolonged low‐intensity cycling on muscular and hormonal responses and recovery in elite cyclists. Twelve male cyclists (VO2max, 73.4 ± 4.0 mL/kg/min) completed a randomized crossover protocol, wherein 4 hours of cycling at 50% of VO2max were performed with and without inclusion of three sets of 3 × 30 seconds maximal sprints (E&S vs E, work‐matched). Muscle biopsies (m. vastus lateralis) and blood were sampled at Pre, immediately after (Post) and 3 hours after (3 h) finalizing sessions. E&S led to greater increases in mRNA levels compared with E for markers of fat metabolism (PDK4, Δ‐Log2 fold change between E&S and E ± 95%CI Post; 2.1 ± 0.9, Δ3h; 1.3 ± 0.7) and angiogenesis (VEGFA, Δ3h; 0.3 ± 0.3), and greater changes in markers of muscle protein turnover (myostatin, ΔPost; −1.4 ± 1.2, Δ3h; −1.3 ± 1.3; MuRF1, ΔPost; 1.5 ± 1.2, all P <.05). E&S showed decreased mRNA levels for markers of ion transport at 3h (Na+‐K+ α1; −0.6 ± 0.6, CLC1; −1.0 ± 0.8 and NHE1; −0.3 ± 0.2, all P <.05) and blunted responses for a marker of mitochondrial biogenesis (PGC‐1α, Post; −0.3 ± 0.3, 3h; −0.4 ± 0.3, P <.05) compared with EE&S and E showed similar endocrine responses, with exceptions of GH and SHBG, where E&S displayed lower responses at Post (GH; −4.1 ± 3.2 μg/L, SHBG; −2.2 ± 1.9 nmol/L, P <.05). Both E&S and E demonstrated complete recovery in isokinetic knee extension torque 24 hours after exercise. In conclusion, we demonstrate E&S to be an effective exercise protocol for elite cyclists, which potentially leads to beneficial adaptations in skeletal muscle without impairing muscle recovery 24 hours after exercise. [ABSTRACT FROM AUTHOR]