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Purpose: In Football, the high‐intensity running bouts during matches are considered decisive. Interestingly, recent studies showed that peak fat oxidation rates (PFO) are higher in football players than other athletes. This study aimed to investigate whether PFO increases following a pre‐season. Secondarily, and due to COVID‐19, we investigated whether PFO is related to the physical performance in a subgroup of semi‐professional male football players. Methods: Before and after 8 weeks of pre‐season training, 42 sub‐elite male football players (18 semi‐professionals and 24 non‐professionals) had a dual‐energy x‐ray absorptiometry scan and performed a graded exercise test on a treadmill for the determination of PFO, the exercise intensity eliciting PFO (Fatmax) and peak oxygen uptake (V̇O2peak). Additionally, the semi‐professional players performed a Yo‐Yo Intermittent Recovery Test level 2 (YYIR2) before and after pre‐season training to determine football‐specific running performance. Results: PFO increased by 11 ± 10% (mean ± 95% CI), p = 0.031, and V̇O2peak increased by 5 ± 1%, p < 0.001, whereas Fatmax was unchanged (+12 ± 9%, p = 0.057), following pre‐season training. PFO increments were not associated with increments in V̇O2peak (Pearson's r2 = 0.00, p = 0.948) or fat‐free mass (FFM) (r2 = 0.00, p = 0.969). Concomitantly, YYIR2 performance increased in the semi‐professional players by 39 ± 17%, p < 0.001, which was associated with changes in V̇O2peak (r2 = 0.35, p = 0.034) but not PFO (r2 = 0.13, p = 0.244). Conclusions: PFO, V̇O2peak, and FFM increased following pre‐season training in sub‐elite football players. However, in a subgroup of semi‐professional players, increments in PFO were not associated with improvements in YYIR2 performance nor with increments in V̇O2peak and FFM. [ABSTRACT FROM AUTHOR]