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Abstract This randomized controlled trial aimed to investigate the effects of eight weeks of lactotripeptide (LTP) ingestion, physical activity (PA) intervention, and combined intervention on the fatigue status of middle-aged and older adults. A total of 78 middle-aged and older adults (63 ± 8 years of age) were randomly assigned to four groups: placebo, LTP, placebo with PA intervention (placebo + PA), and LTP with PA intervention (LTP + PA). All participants ingested the placebo or LTP tablets daily (three tablets/day). The placebo + PA and LTP + PA groups participated in a weekly supervised exercise class and were instructed to increase their moderate- to vigorous-intensity PA at home. The visual analog scale, Brief Fatigue Inventory, Profile of Mood States second edition (POMS2), and Beck Depression Inventory second edition (BDI-II) were administered before and after the intervention. No significant interactions or main effects were observed between LTP ingestion and PA intervention on any of the fatigue scales. The main-effect analyses revealed that the PA intervention improved the total mood disturbance score of the POMS2 (F = 5.22, P = 0.03) and BDI-II score (F = 4.81, P = 0.03). After the post hoc paired comparisons, the total mood disturbance and BDI-II scores improved more with the combined intervention than with the PA intervention alone (percentage difference between the effect of combined intervention and PA intervention alone was 3.7% for total mood disturbance score and 13.7% for BDI-II score). The present study suggests that eight weeks of LTP ingestion and PA intervention did not have a significant effect on fatigue status. However, the PA intervention improved mood status and depressive symptoms, and these effects were enhanced by LTP ingestion.