The use of esophageal recordings of the diaphragm electromyogram (EMG) signal strength to evaluate diaphragm activation during voluntary contractions in humans has recently been criticized because of a possible artifact created by changes in lung volume. Therefore, the first aim of this study was to evaluate whether there is an artifactual influence of lung volume on the strength of the diaphragm EMG during voluntary contractions. The second aim was to measure the required changes in activation for changes in lung volume at a given tension, i.e., the volume-activation relationship of the diaphragm. Healthy subjects (n = 6) performed contractions of the diaphragm at different transdiaphragmatic pressure (Pdi) targets (range 20-160 cmH2O) while maintaining chest wall configuration constant at different lung volumes. The diaphragm EMG was recorded with a multiple-array esophageal electrode, with control of signal contamination and electrode positioning. The effects of lung volume on the EMG were studied by comparing the crural diaphragm EMG root mean square (RMS), an index of crural diaphragm activation, with an index of global diaphragm activation obtained by normalizing Pdi to the maximum Pdi at the given muscle length (Pdi/Pdimax@L) at the different lung volumes. We observed a direct relationship between RMS and Pdi/Pdimax@L independent of diaphragm length. The volume-activation relationship of the diaphragm was equally affected by changes in lung volume as the volume-Pdi relationship (60% change from functional residual capacity to total lung capacity). We conclude that the RMS of the diaphragm EMG is not artifactually influenced by lung volume and can be used as a reliable index of diaphragm activation. The volume-activation relationship can be used to infer changes in the length-tension relationship of the diaphragm at submaximal activation/contraction levels.