In acute hyperinflation, the occurrence of diaphragmatic shortening may alter the contractile characteristics and function of the diaphragm. The aim of this study was to investigate the effects of acute passive diaphragmatic shortening on in vitro mechanical properties and fatigability. Optimal diaphragmatic length (Lo) was defined as being that length at which peak twitch-tension occurred. Acute shortening (85% Lo, 70% Lo) altered the twitch characteristics. At shorter lengths, the time-to-peak tension and the half-relaxation time were significantly reduced (p less than 0.05). These alterations led to marked alterations in the shape of the force-frequency curve at shorter lengths and in the length-tension properties when assessed at different stimulation frequencies. When normalized with respect to maximal tension, a disproportionate decrease in the generated tension was observed at shorter lengths. Fatigability was assessed by repeatedly stimulating the diaphragmatic bundles and observing the drop in tension with respect to time. For a given fatigue regimen, i.e., same stimulation frequency, the shorter diaphragm (70% Lo) generated more absolute force at any given time period. When the initial tensions were matched at Lo and 70% Lo by increasing the stimulation frequency used to fatigue the shorter muscle, the acutely shortened diaphragm generated less absolute force following 60 s of the fatigue regimen. We conclude that alterations in the contractile characteristics of the diaphragm during acute passive shortening are such that a disproportionately greater excitability is required in order to reach a given submaximal tension as at Lo. This factor may partly account for increased diaphragmatic fatiguability in the acutely shortened state.