Pseudomonas aeruginosa can cause acute lung infections in intubated patients or chronic infections in patients with cystic fibrosis (CF). In both situations, P. aeruginosa accumulates specific mutations, in particular in the lasR quorum-sensing regulator gene. Using a Dictyostelium discoideum amoeba model, we assessed whether these mutations affect bacterial virulence. Among a collection of clinical isolates from 16 CF patients, initial isolates were fully virulent in 15 patients, but for late isolates collected several years later, virulence was decreased in eight patients. No significant correlation between genetic inactivation of lasR and decreased virulence was observed. Among strains isolated from ten colonized intubated patients, all initial isolates were fully virulent. Despite the accumulation of lasR-inactivating mutations in strains collected over a 3-week period, no decrease in virulence was observed in eight of 10 patients. In one intubated patient, the virulent initial strain was replaced a few days later with a different, less virulent, strain. We observed a gradual decrease in bacterial virulence in only one intubated patient. We conclude that adaptation of P. aeruginosa to chronically infected CF patients can lead to a slow and gradual loss of virulence, as measured in a Dictyostelium model system. However, loss of virulence is not caused predominantly by mutations in lasR. During short-term colonization of intubated patients for up to 20 days, a decrease in virulence was exceptional, despite the accumulation of lasR mutations.