Measurements of strain and strain rate, obtained by tissue Doppler might provide new parameters for assessing cardiac function in neonates. The noise-to-signal ratio is high. We investigated the effect of the frequency of the probe used, and the settings for tissue Doppler frame rate, on the noise in the analyses in three series of tissue Doppler images. In the first series, we used the 5S probe, with a frequency of 2.4 MHz, and the default frame rate. We used the10S probe, with a frequency of 8.0 MHz, in the other two series, one with a low and one with the default frame rate. The noise was lower using the 5S rather than the 10S probe, and lower when using the low frame rate rather than the default rate with the 10S probe. Using the settings eligible for two segment analyses with the lowest noise for each series, the noise was from 36 to 42% higher when using the 10S probe at default frame rate, and from 13 to 14% higher when using the 10S probe at low frame rate compared to the 5S probe at default frame rate. There were no differences in peak systolic strain or strain rate between the series. We found, therefore, that use of the 5S probe with the default setting for frame rate, along with a length of 1 mm and width of 2 mm for the region of interest, and a strain length of 10 mm, provided the optimal settings for two-segment analyses in this study.