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Acute dietary nitrate (NO 3 −) supplementation can increase [NO 3 −], but not nitrite ([NO 2 −]), in human skeletal muscle, though its effect on [NO 3 −] and [NO 2 −] in skin remains unknown. In an independent group design, 11 young adults ingested 140 mL of NO 3 −-rich beetroot juice (BR; 9.6 mmol NO 3 −), and 6 young adults ingested 140 mL of a NO 3 −-depleted placebo (PL). Skin dialysate, acquired through intradermal microdialysis, and venous blood samples were collected at baseline and every hour post-ingestion up to 4 h to assess dialysate and plasma [NO 3 −] and [NO 2 −]. The relative recovery rate of NO 3 − and NO 2 − through the microdialysis probe (73.1% and 62.8%), determined in a separate experiment, was used to estimate skin interstitial [NO 3 −] and [NO 2 −]. Baseline [NO 3 −] was lower, whereas baseline [NO 2 −] was higher in the skin interstitial fluid relative to plasma (both P < 0.001). Acute BR ingestion increased [NO 3 −] and [NO 2 −] in the skin interstitial fluid and plasma (all P < 0.001), with the magnitude being smaller in the skin interstitial fluid (e.g., 183 ± 54 vs. 491 ± 62 μM for Δ[NO 3 −] from baseline and 155 ± 190 vs. 217 ± 204 nM for Δ[NO 2 −] from baseline at 3 h post BR ingestion, both P ≤ 0.037). However, due to the aforementioned baseline differences, skin interstitial fluid [NO 2 −] post BR ingestion was higher, whereas [NO 3 −] was lower relative to plasma (all P < 0.001). These findings extend our understanding of NO 3 − and NO 2 − distribution at rest and indicate that acute BR supplementation increases [NO 3 −] and [NO 2 −] in human skin interstitial fluid. • [NO 3 −] and [NO 2 −] in skin interstitial fluid was assessed using microdialysis. • Basal [NO 3 −] is lower in skin interstitial fluid relative to plasma. • Basal [NO 2 −] is higher in skin interstitial fluid compared with plasma. • Dietary nitrate (NO 3 −) increases [NO 3 −] and [NO 2 −] in skin interstitial fluid. [ABSTRACT FROM AUTHOR]