The diaphragm is an atypical skeletal muscle in that its length is not determined by changes in joint angle, but rather is influenced by negative intrapleural pressure and movement of the rib cage. In this arrangement, the diaphragm may be susceptible to large changes in sarcomere length thus compromising contractile function and predisposing it to damage. The diaphragmatic ligament may be important in setting muscle length and protecting against muscle damage, particularly during development (1). Therefore, we examined the influence of the diaphragm ligament on contractile function and susceptibility to stretch induced damage in the diaphragm of preterm sheep. This study was approved by the animal ethics committee of the University of Western Australia. Preterm lambs were delivered via hysterotomy at 121 d gestational age (term = 150 d) and killed immediately with pentobarbitone (150 mg/kg IV). The costal diaphragm was dissected and longitudinal strips of muscle fibres were isolated and mounted in an in vitro test system. An adjacent strip of fibres, from which the diaphragm ligament had been carefully removed, was mounted in a second system for concurrent analysis. Muscles were maintained in mammalian Ringer solution (pH 7.3, 25°C) bubbled with carbogen. Each muscle strip was manually adjusted to optimal muscle length (Lo) at which maximum tetanic force (Po), maximum twitch force (Pt), time to peak twitch force (TTP), half relaxation time (½ RT) and maximum rate of force development (df/dt) were determined. Maximum unloaded shortening velocity was determined using the slack test method and susceptibility to muscle damage evaluated from a series of 5 eccentric contractions in which the fibres were stretched to 110% Lo while stimulated. Muscle mass and Lo were recorded for calculation of CSA and specific force (N.cm2). Differences between ligament (LIG: n = 11) and no ligament (NL: n=11) groups were analysed by student t-tests. Removal of the diaphragm ligament had no significant effect on Po or Pt. However, the twitch time course was slower in the NL group as reflected by significant increases (~50%) in TTP and ½ RT and significantly lower df/dt (NL 389 ± 41 g/s, LIG 525 ± 44 g/s; P<0.05). The maximum unloaded shortening velocity was 14% lower in the NL group compared LIG and the decrease in peak force after 5 eccentric contractions was significantly greater without the ligament (NL 4.2 ± 2.2%; LIG 1.2 ± 0.1%; P<0.05). Lo was not significantly different between the groups. These data indicate the importance of the diaphragmatic ligament in maintaining contractile function in the preterm diaphragm and support previous studies suggesting the diaphragmatic ligament provides a structural framework for diaphragm muscle fibre development (2).