Objective: To determine the association between the 5 subscales of the Multidimensional Fatigue Inventory (MFI-20) and physical function in late mid-life. Design: Cross-sectional study. Subjects: A population-based sample of adults who participated in the Copenhagen Aging and Midlife Biobank population cohort (n = 4,964; age 49-63 years). Methods: Self-reported fatigue was measured using the MFI-20 comprising: general fatigue, physical fatigue, reduced activity, reduced motivation, and mental fatigue. Handgrip strength and chair rise tests were used as measures of physical function. Multiple logistic regression analyses were used to determine the associations between handgrip strength and the chair rise test with the MFI-20 subscales, adjusted for potential confounders. Results: After adjustments for potential confounders, handgrip strength was associated with physical fatigue (adjusted odds ratio (OR) 0.75 (95% confidence interval (CI) 0.66-0.86); p ≤ 0.001) and reduced motivation (adjusted OR 0.85 (95% CI 0.75-0.96); p ≤ 0.05), but not with the other subscales. After these adjustments, the chair rise test was associated with physical fatigue (adjusted OR 0.61 (0.53-0.69); p ≤ 0.001), general fatigue (adjusted OR 0.72 (0.62-0.84); p ≤ 0.001), reduced activity (adjusted OR 0.79 (0.70-0.90); p ≤ 0.001) and reduced motivation (adjusted OR 0.84 (0.74-0.95); p ≤ 0.01), but not with mental fatigue. Subgroup analyses for sex did not show statistically significant different associations between physical function and fatigue. Conclusion: The present study supports the physiological basis of 4 subscales of the MFI-20. The association between fatigue and function was independent of gender.