Abstract Background The Pittsburgh Performance Fatigability Index (PPFI) quantifies the percent decline in cadence using accelerometry during standardized walking tasks. Although PPFI has shown strong correlations with physical performance, the developmental sample was relatively homogenous and small, necessitating further validation. Methods Participants from the Study of Muscle, Mobility and Aging (N = 805, age = 76.4 ± 5.0 years, 58% women, 85% White) wore an ActiGraph GT9X on the nondominant wrist during usual-paced 400 m walk. Tri-axial accelerations were analyzed to compute PPFI (higher score = greater fatigability). To evaluate construct and discriminant validity, Spearman correlations (rs) between PPFI and gait speed, Short Physical Performance Battery (SPPB), chair stand speed, leg peak power, VO2peak, perceived fatigability, and mood were examined. Sex-specific PPFI cut-points that optimally discriminated gait speed using classification and regression tree were then generated. Their discriminate power in relation to aforementioned physical performance were further evaluated. Results Median PPFI score was 1.4% (25th–75th percentile range: 0%–21.7%), higher among women than men (p < .001). PPFI score was moderate-to-strongly correlated with gait speed (rs = −0.75), SPPB score (rs = −0.38), chair stand speed (rs = −0.36), leg peak power (rs = −0.34) and VO2peak (rs = −0.40), and less strongly with perceived fatigability (rs = 0.28–0.29), all p < .001. PPFI score was not correlated with mood (|rs| < 0.08). Sex-specific PPFI cut-points (no performance fatigability: PPFI = 0%; mild performance fatigability: 0% < PPFI < 3.5% [women], 0% < PPFI < 5.4% [men]; moderate-to-severe performance fatigability: PPFI ≥ 3.5% [women], PPFI ≥ 5.4% [men]) discriminated physical performance (all p < .001), adjusted for demographics and smoking status. Conclusion Our work underscores the utility of PPFI as a valid measure to quantify performance fatigability in future longitudinal epidemiologic studies and clinical/pharmaceutical trials.