Objective: To determine the effect of changing electrode positions on vital signs and respiratory effort parameters measured with transcutaneous electromyography of the diaphragm (dEMG) in preterm infants. Methods: In this observational study, simultaneous dEMG measurements were performed at the standard position and at one alternative electrode position (randomly assigned to lateral, superior, medial, inferior to the standard placement, or dorsal). The activity of the diaphragm was measured for 1 hour at both positions. Main outcome measures were the agreement in heart rate (HR), respiratory rate (RR), and percentage difference in dEMG parameters of respiratory effort (peak and tonic activity, amplitude, area under the curve, and frequency content) between the standard and alternative electrode positions. Results: Thirty clinically stable preterm infants (gestational age 30.1 ± 3.0 weeks) with either no or noninvasive respiratory support were included. Agreement in HR was excellent at all positions (ICC > 0.95) while RR agreement showed more diversity (ICC range 0.40-0.86). Mixed modeling of dEMG parameters revealed that medial and inferior placement measured the weakest signals (median 75.5% and 64.5% lower dEMG amplitude). Lateral electrode placement showed the highest similarity to standard positioning (median 23.5% lower amplitude). Conclusion: Measuring HR showed high similarity at all positions. However, registration of RR and respiratory effort is clearly influenced by the electrode position. Electrodes in the same transversal plane as the diaphragm, and at sufficient distance from each other, provide the best agreement with the standard positioning.