Diaphragm fatigue was studied in innervated diaphragm strips from 63 Sprague-Dawley rats. The experiments examined 1) the effect on the rate of diaphragmatic fatigue of increases in the diaphragm's duty cycle, i.e., the ratio of the period of diaphragmatic contraction (Ti) to the duration of a cycle of contraction and rest (Ttot) and 2) the possibility that impaired neural transmission contributed to the fatigue process. Alterations in the duty cycle of the diaphragm were simulated by varying the pattern of electrical stimuli applied cyclically to the phrenic nerve. Fatigue was assessed from the rate of fall of isometric tension when the muscle was made to contract 90 times/min. The contribution of neural element fatigue was assessed by comparing the tension during phrenic nerve stimulation to the tension developed when the muscle was stimulated directly. Increasing the duty cycle (Ti/Ttot) from 25 to 50 to 75% increased the rate of diaphragmatic fatigue progressively. Holding Ti/Ttot constant at 75%, while varying Ti and Ttot, did not affect the rate of fatigue. Increases in duty cycle appear to increase the rate of fatigue by increasing the number of times the contractile process was activated. In fatigued muscle strips diaphragmatic tension was greater in directly stimulated muscle than in muscle strips activated via the phrenic nerve. The results indicate that 1) when the breathing action of the diaphragm is simulated in vitro, increases in duty cycle accelerate the fatigue process and 2) failure of transmission of phrenic impulses to diaphragmatic muscle cells contributes to the fall in tension during fatigue.