Accurate assessment of aerobic fitness is crucial in soccer; however, the validity of field-based predictive tests remains uncertain in professional players. This study examined the relationship between directly measured and estimated maximal oxygen uptake (V˙O2max) during the Yo-Yo Endurance Test Level 1 (YYET1) in professional soccer players and evaluated seasonal changes after six months of training and competition. Seventeen players from an Italian third-division team performed the YYET1 in pre- and mid-season conditions, while VO2max was continuously recorded using a portable metabolic system. VO2max was estimated using Bangsbo’s distance-based formula. Linear regression and Bland–Altman analyses were used to assess relationships and agreement between methods. Measured VO2max increased significantly from pre- to mid- season (+13.9%, p < 0.001), whereas estimated values showed a smaller rise (+5.2%, p < 0.001). The predictive method systematically underestimated VO2max (bias −2.3 to −7.0 mL·kg−1·min−1), and regression analyses revealed only moderate shared variance (R2 = 0.18–0.20) between estimated and measured values. These findings demonstrate that Bangsbo’s equation lacks validity for estimating VO2max in professional players and cannot accurately track aerobic adaptations across a season. For precise physiological evaluation, direct measurement using portable metabolic systems is required, while submaximal soccer-specific protocols may offer practical alternatives for longitudinal monitoring.