Objectives: Antiretroviral toxic neuropathy (ATN) is associated with dideoxynucleoside reverse transcriptase inhibitor use in patients infected with HIV, possibly as a result of mitochondrial toxicity. Acetyl-l-carnitine (ALC) has been linked to symptomatic improvement in ATN. We present an open-label single-arm pilot study to evaluate changes in intra-epidermal nerve fibre (IENF) density and mitochondrial DNA (mtDNA) copies/cell among subjects treated with 3000mg ALC daily. Methods: Punch skin biopsies were examined at baseline and after 24 weeks of therapy. Participants reported neuropathic symptoms using the Gracely Pain Intensity Score. Neurological examinations were completed. Results: Twenty-one subjects completed the study. ALC was generally well tolerated. The IENF density did not change in cases completing 24 weeks of ALC therapy, with median (90% confidence interval) IENF changes of -1.70 (-3.50, ∞) (P = 0.98) and 2.15 (-0.10, ∞) (P = 0.11) for the distal leg and proximal thigh, respectively. Fat mtDNA copies/cell did not change with therapy. Improvements in neuropathic pain (P < 0.01), paresthesias (P = 0.01), and symptoms of numbness (P < 0.01) were noted. Similarly, improvement was noted on the Gracely Pain Intensity Score. Conclusions: ALC therapy coincided with improvements in subjective measures of pain in this open-label single-arm study. However, changes were not observed in objective measures of IENF density or mtDNA levels, providing little objective support for use of ALC in this setting.