PURPOSE. The superior and inferior tarsal muscles are sympathetically innervated smooth muscles. Long-term diabetes often leads to microvascular complications, such as, retinopathy and autonomic neuropathy. We hypothesized that diabetes induces (1) sympathetic paresis in the superior and inferior tarsal muscles and that this measure is associated with (2) the severity of diabetic retinopathy, (3) the duration of diabetes, and (4) autonomic function. In addition, association between the severity of retinopathy and autonomic function was investigated. METHODS. Forty-eight participants with long-term type 1 diabetes and confirmed distal symmetrical polyneuropathy were included. Palpebral fissure heights were measured bilaterally in response to topically applied 10% phenylephrine to the right eye. The presence of proliferative diabetic retinopathy (PDR) or nonproliferative diabetic retinopathy and disease duration were denoted. Time and frequency derived heart rate variability parameters obtained from 24-hour continuous electrocardiography were recorded. RESULTS. The difference in palpebral fissure heights between phenylephrine treated and untreated eyes (∆PFH) was 1.02 mm ± 0.29 (P = 0.001). The ∆PFH was significantly lower in the PDR group (0.41 mm ± 0.43 vs. 1.27 mm ± 1.0), F(1,35) = 5.26, P = 0.011. The ∆PFH was lower with increasing diabetes duration, r(37) = −0.612, P = 0.000. Further, the ∆PFH was lower with diminished autonomic function assessed as total frequency power in electrocardiogram (r = 0.417, P = 0.014), and sympathetic measures of very low (r = 0.437, P = 0.010) and low frequency power (r = 0.384, P = 0.025). CONCLUSIONS. The ∆PFH is a simple ambulatory sympathetic measure, which was associated with the presence of PDR, disease duration, and autonomic function. Consequently, ∆PFH could potentially be an inexpensive and sensitive clinical indicator of autonomic dysfunction.