Background: Diaphragm atrophy is observed in mechanically ventilated patients. However, the atrophy is not investigated in other respiratory muscles. Therefore, we conducted a two-center prospective observational study to evaluate changes in diaphragm and intercostal muscle thickness in mechanically ventilated patients. Methods: Consecutive adult patients who were expected to be mechanically ventilated longer than 48 h in the ICU were enrolled. Diaphragm and intercostal muscle thickness were measured on days 1, 3, 5, and 7 with ultrasonography. The primary outcome was the direction of change in muscle thickness, and the secondary outcomes were the relationship of changes in muscle thickness with patient characteristics. Results: Eighty patients (54 males and 26 females; mean age, 68 ± 14 years) were enrolled. Diaphragm muscle thickness decreased, increased, and remained unchanged in 50 (63%), 15 (19%), and 15 (19%) patients, respectively. Intercostal muscle thickness decreased, increased, and remained unchanged in 48 (60%), 15 (19%), and 17 (21%) patients, respectively. Decreased diaphragm or intercostal muscle thickness was associated with prolonged mechanical ventilation (median difference (MD), 3 days; 95% CI (confidence interval), 1-7 and MD, 3 days; 95% CI, 1-7, respectively) and length of ICU stay (MD, 3 days; 95% CI, 1-7 and MD, 3 days; 95% CI, 1-7, respectively) compared with the unchanged group. After adjusting for sex, age, and APACHE II score, they were still associated with prolonged mechanical ventilation (hazard ratio (HR), 4.19; 95% CI, 2.14-7.93 and HR, 2.87; 95% CI, 1.53-5.21, respectively) and length of ICU stay (HR, 3.44; 95% CI, 1.77-6.45 and HR, 2.58; 95% CI, 1.39-4.63, respectively) compared with the unchanged group. Conclusions: Decreased diaphragm and intercostal muscle thickness were frequently seen in patients under mechanical ventilation. They were associated with prolonged mechanical ventilation and length of ICU stay. Trial registration: UMIN000031316. Registered on 15 February 2018.