It is unclear whether physiological responses, such as muscle oxygen saturation (SmO2), can be considered symmetrical during cycling. This knowledge has important practical implications for both training and performance assessment. The aim of this study was to determine whether oxygenation profiles in the three active muscles of both legs were symmetrical during cycling at different intensities. Twenty‐six trained cyclists and triathletes completed a graded exercise test (GXT) and an 8‐min functional threshold power estimation test (8MTT) on a cycle ergometer over two nonconsecutive days. SmO2 was bilaterally assessed using NIRS technology in the vastus lateralis, gastrocnemius medialis, and tibialis anterior. Symmetry was compared between legs in both tests, and reliability and agreement between the measurements were quantified. The main results were that SmO2 in the three muscles assessed did not differ between legs during the GXT and 8MTT (p > 0.05). Reliability of the measures was poor to good in the vastus lateralis (ICC = 0.83–0.37), moderate to excellent in the tibialis anterior (ICC = 0.92–0.73), and poor to good for the gastrocnemius medialis (ICC = 0.80–0.24). Overall, the group variability in SmO2 showed a narrower distribution at lower intensities, with data dispersion increasing at higher intensities. In conclusion, the SmO2 was similar and showed symmetrical responses in both the preferred and nonpreferred limb in different muscles assessed during cycling at different intensities within a range of 10%–20%. Although individual physiological differences that might be relevant in some clinical/performance settings should not be disregarded, these findings indicate that measuring a single lower limb provides an accurate approximation of the responses in both lower limbs.