This study aimed to analyze the variations of fitness status, as well as test the relationships between accumulated training load and fitness changes in women soccer players. This study followed an observational analytic cohort design. Observations were conducted over 23 consecutive weeks (from the preseason to the midseason). Twenty-two women soccer players from the same first Portuguese league team (22.7 ± 5.21 years old) took part in the study. The fitness assessment included anthropometry, hip adductor and abductor strength, vertical jump, change of direction, linear speed, repeated sprint ability, and the Yo-Yo intermittent recovery test. The training load was monitored daily using session rating of perceived exertion (s-RPE). A one-way repeated ANOVA revealed no significant differences for any of the variables analyzed across the three moments of fitness assessments (p > 0.05). The t-test also revealed no differences in the training load across the moments of the season (t = 1.216; p = 0.235). No significant correlations were found between fitness levels and accumulated training load (range: r = 0.023 to −0.447; p > 0.05). This study revealed no differences in the fitness status during the analyzed season, and the fitness status had no significant relationship with accumulated training load.