Abstract Ingestion of sodium nitrate (NO3−) simultaneously reduces whole‐body oxygen consumption (VO2) during sub‐maximal exercise while improving mitochondrial efficiency, suggesting a causal link. Consumption of beetroot juice (BRJ) elicits similar decreases in VO2 but potential effects on the mitochondria remain unknown. Therefore we examined the effects of 7‐day supplementation with BRJ (280 ml d−1, ∼26 mmol NO3−) in young active males (n = 10) who had muscle biopsies taken before and after supplementation for assessments of mitochondrial bioenergetics. Subjects performed 20 min of cycling (10 min at 50% and 70% VO2peak) 48 h before pre (baseline) and post (day 5 of supplementation) biopsies. Whole‐body VO2 decreased (P < 0.05) by ∼3% at 70% VO2peak following supplementation. Mitochondrial respiration in permeabilized muscle fibres showed no change in leak respiration, the content of proteins associated with uncoupling (UCP3, ANT1, ANT2), maximal substrate‐supported respiration, or ADP sensitivity (apparent Km). In addition, isolated subsarcolemmal and intermyofibrillar mitochondria showed unaltered assessments of mitochondrial efficiency, including ADP consumed/oxygen consumed (P/O Ratio), respiratory control ratios (RCR) and membrane potential determined fluorometrically using Safranine‐O. In contrast, rates of mitochondrial hydrogen peroxide (H2O2) emission were increased following BRJ. Therefore, in contrast to sodium nitrate, BRJ supplementation does not alter key parameters of mitochondrial efficiency. This occurred despite a decrease in exercise VO2, suggesting that the ergogenic effects of BRJ ingestion are not due to a change in mitochondrial coupling or efficiency. It remains to be determined if increased mitochondrial H2O2 contributes to this response. This article is protected by copyright. All rights reserved