Purpose Whilst there is evidence to suggest that hypohydration caused by physical work in the heat increases renal injury, whether this is the case during exercise in temperate conditions remains unknown. This study investigated the effect of manipulating hydration status during high-intensity intermittent running on biomarkers of renal injury. Methods After familiarisation, 14 males (age: 33 ± 7 years; V̇O 2peak : 57.1 ± 8.6 ml/kg/min; mean ± SD) completed 2 trials in a randomised cross-over design, each involving 6, 15 min blocks of shuttle running (modified Loughborough Intermittent Shuttle Test protocol) in temperate conditions (22.3 ± 1.0 °C; 47.9 ± 12.9% relative humidity). During exercise, subjects consumed either a volume of water equal to 90% of sweat losses (EU) or 75 mL water (HYP). Body mass, blood and urine samples were taken pre-exercise (baseline/pre), 30 min post-exercise (post) and 24 h post-baseline (24 h). Results Post-exercise, body mass loss, serum osmolality and urine osmolality were greater in HYP than EU ( P  ≤ 0.024). Osmolality-corrected urinary kidney injury molecule-1 (uKIM-1) concentrations were increased post-exercise ( P  ≤ 0.048), with greater concentrations in HYP than EU (HYP: 2.76 1.72–4.65 ng/mOsm; EU: 1.94 1.1–2.54 ng/mOsm; P  = 0.003; median interquartile range). Osmolality-corrected urinary neutrophil gelatinase-associated lipocalin (uNGAL) concentrations were increased post-exercise ( P  < 0.001), but there was no trial by time interaction effect ( P  = 0.073). Conclusion These results suggest that hypohydration produced by high-intensity intermittent running increases renal injury, compared to when euhydration is maintained, and that the site of this increased renal injury is at the proximal tubules.