Despite the positive effects of low-level laser therapy (LLLT) on muscle fatigue before exercises using a single muscle group, the acute effects of LLLT on performance in cardiopulmonary exercise testing (CPET) are poorly understood. We aimed to assess the acute effects of LLLT on physiologic and electromyographic responses to the CPET in healthy adults. A randomized, double-blind, placebo-controlled crossover trial was performed with 18 untrained participants (nine males, 22 ± 2 years). We applied LLLT or placebo on quadriceps and gastrocnemius 10 min before two rapidly incremental CPETs randomly performed in alternate days on a cycle ergometer. Participants received LLLT using a multidiode cluster, 20 s/site (850 nm, 100 mW/diode, 14 J/site). Physiological responses to the CPET were continuously monitored using a gas analyzer. The electromyographic fatigue threshold (EMGth) was assessed through surface electrodes on vastus lateralis. The root mean square (RMS) was plotted every 5 s against the exercise intensity, and its breakpoint values throughout the CPET was identified as EMGth. Compared to placebo, the LLLT significantly increased peak O 2 uptake (V′O 2 33 ± 10 vs. 31 ± 9 mL/min/kg). We observed a shallower slope of the Δheart rate/ΔV′O 2 during the CPET after LLLT compared to placebo, i.e., increased cardiovascular efficiency (56 ± 24 vs. 66 ± 30 bpm/L/min). There were no LLLT-related changes in EMGth. The LLLT acutely increases exercise performance in healthy untrained adults probably due to increased O 2 extraction by peripheral muscles without causing a significant impact on muscle fatigue.