Chronic progressive ophthalmoplegia (CPEO) is a mitochondrial disease. It is part of a spectrum of mitochondrial DNA deletion disorders. In CPEO, by definition, the eye muscles are affected, causing eye movement deficits and ptosis. Phenotypes with further neurologic involvement are often described as CPEO-plus. Other phenotypes within the spectrum are well defined, such as Sensory Ataxia, Neuropathy, Dysarthria and Ophthalmoplegia (SANDO)-syndrome or Kearns-Sayre syndrome. In the future, well defined diagnostic criteria are essential for clinical trials. Diagnostic guidelines are still in development but need validation. However, only few cohorts of patients have been published, so validating clinical data is missing. This study aims to present one of the largest and most elaborate cohorts of patients with CPEO and CPEO-plus. In this retrospective database study, patients with CPEO and CPEO-plus were identified within the Radboudumc, Nijmegen. From clinical records, an extensive list of parameters was collected, including mitochondrial complex deficiencies, pathological DNA mutations and mtDNA deletions, and a full neurologic exam at outpatient clinic visit. Up until 1st of April 2019, 54 patients were included. 46 patients had a CPEO-phenotype, 8 matched CPEO-plus. In 40 patients a pathological genotype was found, of which 34 had mitochondrial DNA mutations or deletions. If present, whole exome sequencing was executed for the other patients to find additional mutations. Ptosis and ophthalmoplegia were the most common clinical manifestations, other manifestations were rare. This study presents one of the largest cohorts with CPEO and CPEO-plus published in literature. The study contributes to the genetic, metabolic, and clinical knowledge of mitochondrial disease. It stresses the need for diagnostic criteria and for well defined clinical outcomes for future clinical trials.