This prospective randomized trial examined the effects of a tripartite intervention (behavioral state modulation + nonnutritive sucking + tucking) on stress from procedural pain during heel pricks. Blood samples for routine screening were collected by heel pricks 48 h after birth (Stage 1) and at ≥37 weeks' gestation (Stage 2); salivary cortisol levels (SCLs) pre-prick (T0) and 20 min post-prick (T1) assessed stress. Preterm infants (n = 64) sampled by convenience at Level III neonatal care units were randomly assigned to the control condition (usual care) or intervention condition (tripartite intervention). Generalized estimating equations examined differences in salivary cortisol between conditions. After adjusting for effects of gestational age, postmenstrual age, and baseline SCLs, (1) at Stage 1, the change in salivary cortisol from T0 to T1 in preterm infants who received the tripartite intervention was, on average, significantly lower by 0.431 units (log scale) than the change in preterm infants who received the control condition (p < 0.001); (2) in the tripartite intervention condition, the difference between the change in mean SCLs from T0 to T1 at Stages 1 and 2 was significantly lower by 0.287 units (log scale), on average than between the change at Stages 1 and 2 in the control condition (p = 0.026). The provision of a tripartite intervention during heel prick significantly decreased the raise of SCLs compared with infants receiving usual care, suggesting lower stress. Clinicians could easily implement the tripartite intervention for heel-stick support; however, replication is needed before recommending its incorporation into routine heel stick and other stressful procedures.