Aim: Spot-tests of urine pH are claimed to be an accessible biomarker of net acid excretion (NAE), and as such, they may be able to determine changes in an individual's intake of acid- or base-forming foods. To test this hypothesis, we aimed to determine if spot-tests of urine pH could index NAE and relay the consumption of a fruit and vegetable (F&V) concentrate whilst determining this concentrate's capacity to modulate NAE. Methods: In a double blind, placebo-controlled, cross-over trial, healthy adults (n = 13) were allocated by simple randomisation to receive a F&V concentrate or placebo for three days each, with diet standardised throughout. Measurements of 24-hour NAE, 24-hour urine pH and spot-tests of urine pH were taken throughout the study. Results: The 24-hour urine pH predicted 24-hour NAE (P = <0.0001). However, spot-tested urine pH displayed prediction intervals too wide to infer 24-hour NAE and inconsistent ability to reflect concentrate ingestion, despite 24-hour NAE and 24-hour urine pH decreasing (−25.8 mEq, 95% CI −44.3 to −7.4, P = 0.01, d = 0.94) and increasing (+0.51, 95% CI 0.25–0.79, P = 0.002, d = 1.3), respectively, following supplementation. Conclusions: Spot-tests of urine pH are not a valid dietary biomarker of daily NAE and were unable to reliably track changes, despite a F&V concentrate clearly modulating the daily rate of NAE.