This prospective cohort study aimed to evaluate whether COVID-19 lockdown caused biological, psychological, and/or physical performance variations in footballers. We compared the 2018/2019 and 2019/2020 seasons evaluating the plasma volume, hematological parameters, iron/ferritin, creatine kinase, vitamin D, cortisol, testosterone, and physiological state of players of the Italian football major league (Serie A). Measurements were performed before the preparatory period (T0), at the beginning (T1) and in the middle (T2) of the championship, and in March (T3) and at the end of season (T4). The results showed that in the 2019/2020 season affected by the lockdown, the weight, BMI, and fat mass percentage were higher than in the previous season. Hematocrit, hemoglobin, red blood cells, and ferritin decreased during both seasons, more significantly than in the regular season. During both seasons, creatine kinase increased from T2 whilst iron concentrations decreased in T3. Testosterone increased in both seasons from T0 to T3 and returned to initial levels at T4; cortisol increased in T2 and T3 during the 2018/2019 season but not during the COVID-19 season. Physical performance tests revealed differences associated with lockdown. Thus, although from a medical point of view, none of the evaluated changes between the two seasons were clinically relevant, training at home during lockdown did not allow the players to maintain the jumping power levels typical of a competitive period.