Purpose To evaluate the relationship of neuroretinal layer thickness with sensitive measures of cardiovascular autonomic neuropathy in diabetic patients with non‐proliferative diabetic retinopathy (NPDR). Methods Twenty‐seven eyes of 27 patients with type 1 diabetes presenting with mild‐to‐moderate NPDR were compared to 27 healthy control (HC) eyes matched for age and gender. The total macular volume (TMV) and the volumes of individual neurosensory layers in the macula were analysed from spectral domain optical coherence tomography using automated layer segmentation. Cardiovascular autonomic regulation was assessed by short‐term power spectrum analysis of heart rate variability (HRV) before, during and after an orthostatic challenge. Results The patients had an age of 46 ± 12 years and diabetes since 28 ± 9 years. Diastolic and mean arterial pressure was lower in the patients compared to HCs. TMV (r = 0.58, p = 0.002), inner plexiform layer volume (IPLV; r = 0.39, p = 0.047) and inner nuclear layer volume (INLV; r = 0.60, p = 0.001) were associated with reduced recovery of low‐frequency (LF) spectral power of HRV after orthostatic load in diabetic patients but not in HCs. The response of LF spectral power during the orthostatic manoeuvre was blunted in patients compared to HCs (p = 0.02). Diabetes duration was negatively associated with TMV and INLV, whereas IPLV was significantly reduced in eyes with moderate NPDR compared to HCs. Conclusion The results indicate a correlation between inner retinal tissue loss and diminished autonomic regulation in type 1 diabetic patients with mild‐to‐moderate NPDR. The observed changes can be interpreted as congruent early signs of retinal and systemic neuropathy in diabetes.